AU2007246772B2 - Polymorphisms in growth hormone receptor, ghrelin, leptin, neuropeptide Y, and uncoupling protein 2 genes and their associations with measures of performance and carcass merit in beef cattle - Google Patents

Polymorphisms in growth hormone receptor, ghrelin, leptin, neuropeptide Y, and uncoupling protein 2 genes and their associations with measures of performance and carcass merit in beef cattle Download PDF

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AU2007246772B2
AU2007246772B2 AU2007246772A AU2007246772A AU2007246772B2 AU 2007246772 B2 AU2007246772 B2 AU 2007246772B2 AU 2007246772 A AU2007246772 A AU 2007246772A AU 2007246772 A AU2007246772 A AU 2007246772A AU 2007246772 B2 AU2007246772 B2 AU 2007246772B2
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animals
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animal
ghr
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Stephen Moore
Joshua Donald Nkrumah
Esther Laura Sherman
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University of Alberta
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16HHEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H70/00ICT specially adapted for the handling or processing of medical references
    • G16H70/60ICT specially adapted for the handling or processing of medical references relating to pathologies
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/124Animal traits, i.e. production traits, including athletic performance or the like
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/172Haplotypes

Abstract

The physiological regulation of intake, growth and energy partitioning in animals is under the control of multiple genes, which may be important candidates for unraveling the genetic variation in economically relevant traits in beef production. The present invention relates to the identification of a single nucleotide polymorphisms (SNPs) within the bovine genes encoding growth hormone receptor (GHR), ghrelin, leptin, neuropeptide Y (NPY), and Uncoupling Protein 2 (UCP2) and their association with economically relevant traits in beef production. The invention further encompasses methods and systems, including network- based processes, to manage the SNP data and other data relating to specific animals and herds of animals, veterinarian care, diagnostic and quality control data and management of livestock which, based on genotyping, have predictable meat quality traits, husbandry conditions, animal welfare, food safety information, audit of existing processes and data from field locations.

Description

WO 2007/129219 PCT/IB2007/001717 1 TITLE OF THE INVENTION POLYMORPHISMS IN GROWTH HORMONE RECEPTOR, GHRELIN, LEPTIN, NEUROPEPTIDE Y, AND UNCOUPLING PROTEIN 2 GENES AND THEIR ASSOCIATIONS WITH MEASURES OF PERFORMANCE AND CARCASS MERIT IN BEEF CATTLE 5 INCORPORATION BY REFERENCE This application claims benefit of U.S. provisional patent application Serial Nos. 60/758,616 filed January 13, 2006 and 60/836,777 filed August 10, 2006. The foregoing applications, and all documents cited therein or during their 10 prosecution ("appln cited documents"). and all documents cited or referenced in the appln cited documents, and all documents cited.or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby 15 incorporated herein by reference, and may be employed in the practice of the invention. FIELD OF THE INVENTION The present invention relates to the identification of a single nucleotide polymorphisms (SNPs) within the bovine genes encoding growth hormone receptor (GHR), ghrelin, leptin, neuropeptide Y (NPY), and Uncoupling Protein 2 (UCP2) and their 20 association with economically relevant traits in beef production. The invention further relates to methods and systems, including network-based processes, to manage the SNP data and other data relating to specific animals and herds of animals, veterinarian care, diagnostic and quality control data and management of livestock which, based on genotyping, have predictable meat quality traits, husbandry conditions, animal welfare, food safety 25 information, audit of existing processes and data from field locations. BACKGROUND OF THE INVENTION Significant improvements in animal performance, efficiency and carcass and meat quality have been made over the years through the application of standard animal breeding and selection techniques. However, such classical animal breeding techniques require several 30 years of genetic evaluation of performance records on individual animals and their relatives and are therefore very expensive. Other efforts have been made to improve productivity and quality through the application of such management practices as the use of feed additives, animal hormonal implants and chemotherapeutics. However, there is significant political and regulatory resistance to the introduction and use of such methodologies. Such methodologies 35 are also non-inheritable and need to be applied differently in every production system.
WO 2007/129219 PCT/IB2007/001717 2 There is a need for methods that allow relatively easy and more efficient selection and breeding of farm animals with an advantage for an inheritable trait of circulating leptin levels, feed intake, growth rate, body weight, carcass merit and carcass composition. The economic significance of the use of genetic markers that are associated with specific economically 5 important traits (especially traits with low heritability) in livestock through marker-assisted selection cannot therefore be over-emphasized. The physiological regulation of intake, growth and energy partitioning in animals is under the control of multiple genes, which may be important candidates for unraveling the genetic variation in economically relevant traits (ERT) in beef production. Polymorphisms in 10 these candidate genes that show association with specific ERT are useful quantitative trait nucleotides for marker-assisted selection. In the present study, associations between single nucleotide polymorphisms (SNPs) in the bovine growth hormone receptor (GHR), bovine neuropeptide Y (NPY), leptin, ghrelin and uncoupling protein 2 (UCP2) genes with measures of intake, growth and carcass merit in beef cattle. 15 The GHR is bound by GH in a homodimeric group resulting in the initiation of signal transduction mechanisms and the subsequent activation of many hormonal systems involved in growth promotion as well as lipid, nitrogen, mineral and carbohydrate metabolism. The interactions between GH and its receptor also affect protein synthesis, protein degradation, and regulation of overall protein turnover. Other areas of activity are effects on nitrogen 20 retention, fat synthesis, fatty acid oxidation, and stimulation of fatty acid mobilization from body adipose tissues. Treatment of farm animals with growth hormone has been shown to lead to decreased feed intake, increased average daily gain, increased feed efficiency, decreased fat accretion and increased protein accretion. Ghrelin is a growth hormone releasing peptide, consisting of 28-amino acids, which 25 serves as an endogenous ligand for growth hormone-secretagogue (G-protein-coupled). These receptors in turn stimulate the release of GH from the pituitary gland. In addition, ghrelin has also been shown to play important roles in the stimulation of appetite and feeding activity through interactions with peptides such as NPY. Leptin, the hormone product of the ob (obese) gene, has been shown to be 30 predominantly synthesized and expressed in adipose tissues. It functions as a potent physiological signal in the regulation of body weight, energy expenditure, feed intake, adiposity, fertility and immune functions. Leptin has been proposed as one of the major control factors contributing to the phenotypic and genetic variation in the performance and efficiency of cattle.
WO 2007/129219 PCT/IB2007/001717 3 Polymorphisms in the coding regions of the leptin gene in cattle have been associated with milk yield and composition (see, e.g., Liefers et al., J Dairy Sci. 2002 Jun;85(6):1633-8), feed intake (see, e.g., Liefers et al., J Dairy Sci. 2002 Jun;85(6):1633-8; Lagonigro et al., Anim Genet. 2003 Oct;34(5):371-4), and body fat (see, e.g., Buchanan et al., Genet Sel Evol. 5 2002 Jan-Feb;34(1):105-16; Lagonigro et al., Anim Genet. 2003 Oct;34(5):371-4). Polymorphisms in the leptin promoter have been identified, specifically the UASMS 1, UASMS2, UASMS3, E2JW, and E2FB SNPs (see, e.g., Nkrumah et al., J Anim Sci. 2005 Jan;83(l):20-8; Schenkel et al., J Anim Sci. 2005 Sep;83(9):2009-20) and the A59V SNP (see, e.g., Liefers et al., Mamm Genome. 2003 Sep;14(9):657-63), however, only the UASM2 10 SNP (see, e.g., Nkrumah et al., J Anim Sci. 2005 Jan;83(1):20-8) has been associated with serum leptin concentration and economically relevant traits of growth, feed intake, efficiency and carcass merit in cattle. Neuropeptide Y is a 36-amino acid peptide that plays a powerful role as a central appetite stimulator playing important roles in the regulation and control of food intake and 15 energy-balance. It stimulates food intake and induces a general anabolic state by reducing energy expenditure. Additionally, NPY influences the regulation of growth in animals by causing a dose-dependent inhibition of GH release, and a lowering of plasma GH and IGF-l concentration through the stimulation of somatostatin. Uncoupling proteins are proteins that can uncouple ATP production from 20 mitochondrial respiration, by causing a proton leakage, leading to the dissipation of energy as heat. Although certain uncoupling proteins have been shown to influence variations in metabolic efficiency and thermogenesis, the role of UCP2 in energy balance is currently unclear. Nevertheless, UCP2 has been shown to regulate insulin secretion, and it is up regulated by a high-fat diet, suggesting UCP2 to be important for determining basal 25 metabolic rate-and possibly resistance to obesity. Most importantly, significant genetic linkage has been established between microsatellite markers encompassing the location of UCP2 with resting metabolic rate, body mass, body fatness and fat mass in humans. It remains advantageous to provide further SNPs that may more accurately predict the meat quality phenotype of an animal and also a business method that provides for increased 30 production efficiencies in livestock cattle, as well as providing access to various records of the animals and allows comparisons with expected or desired goals with regard to the quality and quantity of animals produced. Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
WO 2007/129219 PCT/IB2007/001717 4 SUMMARY OF THE INVENTION The present invention relates to the identification of a single nucleotide polymorphisms (SNPs) within the bovine genes encoding growth hormone receptor (GHR), ghrelin, leptin, neuropeptide Y (NPY), and Uncoupling Protein 2 (UCP2) and their 5 association with economically relevant traits in beef production. The invention encompasses a method for sub grouping animals according to genotype wherein the animals of each sub-group have a similar polymorphism in a GHR, ghrelin, leptin, NPY or UCP2 gene which may comprise determining the genotype of each animal to be subgrouped by determining the presence of a single nucleotide polymorphism in the GHR, 10 ghrelin, leptin, NPY or UCP2 gene, and segregating individual animals into sub-groups wherein each animal in a subgroup has a similar polymorphism in the GHR, ghrelin, leptin, NPY or UCP2 gene. The invention also encompasses a method for sub grouping animals according to genotype wherein the animals of each sub-group have a similar genotype in the GHR, 15 ghrelin, leptin, NPY or UCP2 gene which may comprise determining the genotype of each animal to be subgrouped by determining the presence of a single nucleotide polymorphism(s) of interest in the GHR, ghrelin, leptin, NPY or UCP2 gene, and segregating individual animals into sub-groups depending on whether the animals have, or do not have, the single nucleotide polymorphism(s) of interest in the GHR, ghrelin, leptin, NPY or UCP2 gene. 20 The single nucleotide polymorphism(s) of interest may be selected from the group consisting of an A to G substitution at the 300 nucleotide position in intron 4 of the GHR gene, an A to G substitution at position 212 in intron 3 of the ghrelin gene, a C to T mutation at position 528 in the leptin gene, a C to T mutation at position 321 in the leptin gene, an A to G substitution at the 666 nucleotide position in intron 2 of the NPY gene, an A to G 25 substitution at position 812 of exon 4 in the UCP2 gene and a C to G substitution at position 213 in intron 2 of the UCP2 gene. The invention further relates to a method for sub grouping animals according to genotype wherein the animals of each sub-group have a similar genotype in the GHR, ghrelin,. leptin, NPY or UCP2 gene which may comprise determining the genotype of each 30 animal to be subgrouped by determining the presence of any one of the above SNPs, and segregating individual animals into sub-groups depending on whether the animals have, or do not have, any one of the above SNPs in the GHR, ghrelin, leptin, NPY or UCP2 gene. The invention also relates to method for identifying an animal having a desirable phenotype relating to certain feed intake, growth rate, body weight, carcass merit and 5 composition, and milk yield, as compared to the general population of animals of that species, which may comprise determining the presence of a single nucleotide polymorphism in the GHR, ghrelin, leptin, NPY or UCP2 gene of the animal, wherein the presence of the SNP is indicative of a desirable phenotype relating to certain feed 5 intake, growth rate, body weight, carcass merit and composition, and milk yield. The invention further relates to a method for identifying a bovine animal having a higher average daily gain (ADG), a higher final weight (FW), a higher dry matter intake (DMI), a higher metabolic mid-weight (MMW), a higher slaughter weight (SW), a higher loin muscle area (LMA), a higher ultrasound loin muscle area (ULMA), and a 10 higher feed conversion ratio (FCR) as compared to a general population of bovine animals, comprising the steps of: (a) obtaining a biological sample from said bovine animal, wherein the sample comprises nucleic acids comprising the bovine growth hormone receptor (GHR) gene; 15 (b) detecting in said nucleic acids the presence of a single nucleotide polymorphism (SNP) in the GHR gene, wherein the SNP is a G in both alleles of the GHR gene at position corresponding to position 300 of SEQ ID NO: 1; and (c) correlating the G in both alleles of the GHR gene to higher FW, DMI, MMW, SW, LMA, ULMA, and FCR, thereby identifying said bovine animal. 20 In an advantageous embodiment, the animal may be a bovine. In another advantageous embodiment, the GHR, ghrelin, leptin, NPY or UCP2 gene may be a bovine GHR, ghrelin, leptin, NPY or UCP2 gene. The invention also encompasses computer-assisted methods and systems for improving the production efficiency for livestock having marketable tender meat using 25 multiple data, and in particular the genotype of the animals as it relates to GHR, ghrelin, leptin, NPY or UCP2 SNPs. Methods of the invention encompass obtaining a genetic sample from each animal in a herd of livestock, determining the genotype of each animal with respect to specific quality traits as defined by a panel of at least two single polynucleotide polymorphisms (SNPs), grouping animals with like genotypes, 30 and optionally, further sub-grouping animals based on like phenotypes. Methods of the invention may also encompass obtaining and maintaining data relating to the animals or to herds, their husbandry conditions, health and veterinary care and condition, genetic history or parentage, and providing this data to others through systems that are web based, contained in a database, or attached to the animal itself such as by an implanted 35 microchip. An advantageous aspect of the present invention, therefore, is directed to a 6 computer system and computer-assisted methods for tracking quality traits for livestock possessing specific genetic predispositions. The present invention advantageously encompasses computer-assisted methods and systems for acquiring genetic data, particularly genetic data as defined by the 5 absence or presence of a SNP within the GHR, ghrelin, leptin, NPY or UCP2 gene related to meat quality traits of the breed of animal and associating that data with other data about the animal or its herd, and maintaining that data in ways that are accessible. Another aspect of the invention encompasses a computer-assisted method for predicting which livestock animals possess a biological difference in meat quality, and 10 which may include the steps of using a computer system, e.g., a programmed computer comprising a processor, a data storage system, an input device and an output device, the steps of: (a) inputting into the programmed computer through the input device data that includes a genotype of an animal as it relates to any one of the GHR, ghrelin, leptin, NPY or UCP2 SNPs described herein, (b) correlating meat quality predicted by the 15 GHR, ghrelin, leptin, NPY or UCP2 genotype using the processor and the data storage system and (c) outputting to the output device the meat quality correlated to the GHR, ghrelin, leptin, NPY or UCP2 genotype, thereby predicting which livestock animals possess a particular meat quality. Yet another aspect of the invention relates to a method of doing business for 20 managing livestock comprising providing to a user computer system for managing livestock comprising physical characteristics and genotypes corresponding to one or more animals or a computer readable media for managing livestock comprising physical characteristics and genotypes corresponding to one or more animals or physical characteristics and genotypes corresponding to one or more animals, wherein a 25 physical characteristic intake, growth or carcass merit in beef cattle and the genotype is a GHR, ghrelin, leptin, NPY or UCP2 genotype. The invention further relates to a method for producing a population of bovine animals having a higher number of offspring with a higher average daily gain (ADG), a higher final weight (FW), a higher dry matter intake (DMI), a higher metabolic mid 30 weight (MMW), a higher slaughter weight (SW), a higher loin muscle area (LMA), a higher ultrasound loin muscle area (ULMA), and a higher feed conversion ratio (FCR), as compared to a general population of bovine animals, comprising the steps of: (a) obtaining a biological sample from each bovine animal from a general population of bovine animals, wherein the sample comprises nucleic acids comprising 35 the bovine growth hormone receptor (GHR) gene; 6A (b) detecting in said nucleic acids the presence of a single nucleotide polymorphism (SNP) in the GHR gene, wherein the SNP is a G in both alleles of the GHR gene at position corresponding to position 300 of SEQ ID NO: 1; (c) segregating individual bovine animals into sub-groups depending on 5 whether the animals have, or do not have, a G in either or both alleles of the GHR gene at position corresponding to position 300 of SEQ ID NO:1; (d) continuously breeding only animals that are heterozygous or homozygous for the G in the GHR gene at position corresponding to position 300 of SEQ ID NO:1, thereby producing the population of bovine animals. 10 It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of' and "consists essentially of' have the meaning ascribed to them in U.S. Patent law, e.g., 15 they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general 20 knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application. These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description. 25 BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which: FIG. I depicts the nucleotide sequence of a GHR gene (Accession No. 30 AY643807, species, bos taurus), SEQ ID NO: 1; FIG. 2 depicts the nucleotide sequence of a bovine leptin promoter (GenBank accession no. AB070368, species, bos taurus), SEQ ID NO: 2; FIG. 3 depicts the nucleotide sequence of a bovine leptin promoter (GenBank accession no. BTA512639; EMBL Accession no. AJ512639), SEQ ID NO: 3; 35 FIG. 4 depicts the nucleotide sequence of intron 3 of the bovine ghrelin gene (unpublished) SEQ ID NO: 4 with a SNP indicated in brackets; 6B FIG. 5 depicts the nucleotide sequence of a bovine leptin promoter (intron 2 of the NPY gene (Accession No. AY491054, species bos taurus), SEQ ID NO: 5; WO 2007/129219 PCT/IB2007/001717 7 FIG. 6 depicts the nucleotide sequence of exon 4 of the bovine UCP2 gene (Accession No. XM_614452, species bos taurus), SEQ ID NO: 6; FIG. 7 depicts the nucleotide sequence of intron 2 of the UCP2 gene (unpublished) SEQ ID NO: 4 with a SNP indicated in brackets; and 5 FIG. 8 illustrates the associations of SNP genotypes with final weight of beef steers (least square means ± SE). Significant differences between genotypes of the SNP denoted as: *P <0.05, ** P < 0.01, *** P < 0.001, and no * P<0.10. FIG. 9 illustrates a flowchart of the input of data and the output of results from the analysis and correlation of the data pertaining to the breeding, veterinarian histories and 10 performance requirements of a group of animals such as from a herd of cows and the interactive flow of data from the computer-assisted device to a body of students learning the use of the method of the invention. FIG. 10 illustrates potential relationships between the data elements to be entered into the system. Unidirectional arrows indicate, for example, that a house or shed is typically 15 owned by only one farm, whereas a farm may own several houses or sheds. Similarly, a prescription may include have several veterinarian products. FIG. I 1A illustrates the flow of events in the use of the portable computer-based system for data entry on the breeding and rearing of a herd of cows. FIG. 11 B illustrates the flow of events through the sub-routines related to data entry 20 concerning farm management. FIG. 11 C illustrates the flow of events through the sub-routines related to data entry concerning data specific to a company. FIG. 12 illustrates a flow chart of the input of data and the output of results from the analysis and the correlation of the data pertaining to the breeding, veterinarian histories, and 25 performance requirements of a group of animals. DETAILED DESCRIPTION The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in 30 the literature. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press; DNA Cloning, Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cells WO 2007/129219 PCT/IB2007/001717 8 and Enzymes (IRL press, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications). 5 Before describing the present invention in detail, it is to be understood that this invention is not limited to particular DNA, polypeptide sequences or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. 10 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein. 15 In describing the present invention, the following terms will be employed and are intended to be defined as indicated below. The term "cow" or "cattle" is used generally to refer to an animal of bovine origin of any age. Interchangeable terms include "bovine", "calf', "steer", "bull", "heifer" and the like. It also includes an individual animal in all stages of development, including embryonic 20 and fetal stages. The animals as referred to herein may also include individuals or groups of individuals that are raised for other than food production such as, but not limited to, transgenic animals for the production of biopharmaceuticals including antibodies and other proteins or protein products. By the term "complementarity" or "complementary" is meant, for the purposes of the 25 specification or claims, a sufficient number in the oligonucleotide of complementary base pairs in its sequence to interact specifically (hybridize) with a target nucleic acid sequence of the gene polymorphism to be amplified or detected. As known to those skilled in the art, a very high degree of complementarity is needed for specificity and sensitivity involving hybridization, although it need not be 100%. Thus, for example, an oligonucleotide that is 30 identical in nucleotide sequence to an oligonucleotide disclosed herein, except for one base change or substitution, may function equivalently to the disclosed oligonucleotides. A "complementary DNA" or "cDNA" gene includes recombinant genes synthesized by reverse transcription of messenger RNA ("mRNA"). A "cyclic polymerase-mediated reaction" refers to a biochemical reaction in which a WO 2007/129219 PCT/IB2007/001717 9 template molecule or a population of template molecules is periodically and repeatedly copied to create a complementary template molecule or complementary template molecules, thereby increasing the number of the template molecules over time. By the term "detectable moiety" is meant, for the purposes of the specification or 5 claims, a label molecule (isotopic or non-isotopic) which is incorporated indirectly or directly into an oligonucleotide, wherein the label molecule facilitates the detection of the oligonucleotide in which it is incorporated, for example when the oligonucleotide is hybridized to amplified gene polymorphisms sequences. Thus, "detectable moiety" is used synonymously with "label molecule". Synthesis of oligonucleotides can be accomplished by 10 any one of several methods known to those skilled in the art. Label molecules, known to those skilled in the art as being useful for detection, include chemiluminescent, fluorescent or luminescent molecules. Various fluorescent molecules are known in the art which are suitable for use to label a nucleic acid for the method of the present invention. The protocol for such incorporation may vary depending upon the fluorescent molecule used. Such 15 protocols are known in the art for the respective fluorescent molecule. "DNA amplification" as used herein refers to any process that increases the number of copies of a specific DNA sequence by enzymatically amplifying the nucleic acid sequence. A variety of processes are known. One of the most commonly used is the polymerase chain reaction (PCR) process of Mullis as described in U.S. Pat. Nos. 4,683,195 and 4,683,202. 20 Methods, devices and reagents as described in U.S. Patent Nos. 6,951,726; 6,927,024; 6,924,127; 6,893,863; 6,887,664; 6,881,559; 6,85.5,522; 6,855,521; 6,849,430; 6,849,404; 6,846,631; 6,844,158; 6,844,155; 6,818,437; 6,818,402; 6,794,177; 6,794,133; 6,790,952; 6,783,940; 6,773,901; 6,770,440; 6,767,724; 6,750,022; 6,744,789; 6,733,999; 6,733,972; 6,703,236; 6,699,713; 6,696,277; 6,664,080; 6,664,064; 6,664,044; RE38,352; 6,650,719; 25 6,645,758; 6,645,720; 6,642,000; 6,638,716; 6,632,653; 6,617,107; 6,613,560; 6,610,487; 6,596,492; 6,586,250; 6,586,233; 6,569,678; 6,569,627; 6,566,103; 6,566,067; 6,566,052; 6,558,929; 6,558,909; 6,551,783; 6,544,782; 6,537,752; 6,524,830; 6,518,020; 6,514,750; 6,514,706; 6,503,750; 6,503,705; 6,493,640; 6,492,114; 6,485,907; 6,485,903; 6,482,588; 6,475,729; 6,468,743; 6,465,638; 6,465,637; 6,465,171; 6,448,014; 6,432,646; 6,428,987; 30 6,426,215; 6,423,499; 6,410,223; 6,403,341; 6,399,320; 6,395,518; 6,391,559; 6,383,755; 6,379,932; 6,372,484; 6,368,834; 6,365,375; 6,358,680; 6,355,422; 6,348,336; 6,346,384; 6,319,673; 6,316,195; 6,316,192; 6,312,930; 6,309,840; 6,309,837; 6,303,343; 6,300,073; 6,300,072; 6,287,781; 6,284,455; 6,277,605; 6,270,977; 6,270,966; 6,268,153; 6,268,143; D445,907; 6,261,431; 6,258,570; 6,258,567; 6,258,537; 6,258,529; 6,251,607; 6,248,567; WO 2007/129219 PCT/IB2007/001717 10 6,235,468; 6,232,079; 6,225,093; 6,221,595; D441,091; 6,218,153; 6,207,425; 6,183,999; 6,183,963; 6,180,372; 6,180,349 ; 6,174,670; 6,153,412; 6,146,834; 6,143,496; 6,140,613; 6,140,110; 6,103,468; 6,087,097; 6,072,369; 6,068,974; 6,063,563; 6,048,688; 6,046,039; 6,037,129; 6,033,854; 6,031,960; 6,017,699; 6,015,664; 6,015,534; 6,004,747; 6,001,612; 5 6,001,572; 5,985,619; 5,976,842; 5,972,602; 5,968,730; 5,958,686; 5,955,274; 5,952,200; 5,936,968; 5,909,468; 5,905,732; 5,888,740; 5,883,924; 5,876,978; 5,876,977; 5,874,221; 5,869,318; 5,863,772; 5,863,731; 5,861,251; 5,861,245; 5,858,725; 5,858,718; 5,856,086; 5,853,991; 5,849,497; 5,837,468; 5,830,663; 5,827,695; 5,827,661; 5,827,657; 5,824,516; 5,824,479; 5,817,797; 5,814,489; 5,814,453; 5,811,296; 5,804,383; 5,800,997; 5,780,271 10 5,780,222; 5,776,686; 5,774,497; 5,766,889; 5,759,822; 5,750,347; 5,747,251; 5,741,656; 5,716,784; 5,712,125; 5,712,090; 5,710,381; 5,705,627; 5,702,884; 5,693,467; 5,691,146; 5,681,741; 5,674,717; 5,665,572; 5,665,539; 5,656,493; 5,656,461; 5,654,144; 5,652,102; 5,650,268; 5,643,765; 5,639,871; 5,639,611; 5,639,606; 5,631,128; 5,629,178; 5,627,054; 5,618,703; 5,618,702; 5,614,388; 5,610,017; 5,602,756; 5,599,674; 5,589,333; 5,585,238; 15 5,576,197; 5,565,340; 5,565,339; 5,556,774; 5,556,773; 5,538,871; 5,527,898; 5,527,510; 5,514,568; 5,512,463; 5,512,462; 5,501,947; 5,494,795; 5,491,225; 5,487,993; 5,487,985; 5,484,699; 5,476,774; 5,475,610; 5,447,839; 5,437,975; 5,436,144; 5,426,026; 5,420,009; 5,411,876; 5,393,657; 5,389,512; 5,364,790; 5,364,758; 5,340,728; 5,283,171; 5,279,952; 5,254,469; 5,241,363; 5,232,829; 5,231,015; 5,229,297; 5,224,778; 5,219,727; 5,213,961; 20 5,198,337; 5,187,060; 5,142,033; 5,091,310; 5,082,780; 5,066,584; 5,023,171 and 5,008,182 may also be employed in the practice of the present invention. PCR involves the use of a thermostable DNA polymerase, known sequences as primers, and heating cycles, which separate the replicating deoxyribonucleic acid (DNA), strands and exponentially amplify a gene of interest. Any type of PCR, such as quantitative PCR, RT-PCR, hot start PCR, 25 LAPCR, multiplex PCR, touchdown PCR, etc., may be used. Advantageously, real-time PCR is used. In general, the PCR amplification process involves a cyclic enzymatic chain reaction for preparing exponential quantities of a specific nucleic acid sequence. It requires a small amount of a sequence to initiate the chain reaction and oligonucleotide primers that will hybridize to the sequence. In PCR the primers are annealed to denatured nucleic acid 30 followed by extension with an inducing agent (enzyme) and nucleotides. This results in newly synthesized extension products. Since these newly synthesized sequences become templates for the primers, repeated cycles of denaturing, primer annealing, and extension results in exponential accumulation of the specific sequence being amplified. The extension product of the chain reaction will be a discrete nucleic acid duplex with a termini WO 2007/129219 PCT/IB2007/001717 11 corresponding to the ends of the specific primers employed. By the terms "enzymatically amplify" or "amplify" is meant, for the purposes of the specification or claims, DNA amplification, i.e., a process by which nucleic acid sequences are amplified in number. There are several means for enzymatically amplifying nucleic acid 5 sequences. Currently the most commonly used method is the polymerase chain reaction (PCR). Other amplification methods include LCR (ligase chain reaction) which utilizes DNA ligase, and a probe consisting of two halves of a DNA segment that is complementary to the sequence of the DNA to be amplified, enzyme QB replicase and a ribonucleic acid (RNA) sequence template attached to a probe complementary to the DNA to be copied which is used 10 to make a DNA template for exponential production of complementary RNA; strand displacement amplification (SDA); QB replicase amplification (QBRA); self-sustained replication (3SR); and NASBA (nucleic acid sequence-based amplification), which can be performed on RNA or DNA as the nucleic acid sequence to be amplified. A "fragment" of a molecule such as a protein or nucleic acid is meant to refer to any 15 portion of the amino acid or nucleotide genetic sequence. As used herein, the term "genome" refers to all the genetic material in the chromosomes of a particular organism. Its size is generally given as its total number of base pairs. Within the genome, the term "gene" refers to an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional 20 product (e.g., a protein or RNA molecule). In general, an animal's genetic characteristics, as defined by the nucleotide sequence of its genome, are known as its "genotype," while the animal's physical traits are described as its "phenotype." By "heterozygous" or "heterozygous polymorphism" is meant that the two alleles of a diploid cell or organism at a given locus are different, that is, that they have a different 25 nucleotide exchanged for the same nucleotide at the same place in their sequences. By "homozygous" or "homozygous polymorphism" is meant that the two alleles of a diploid cell or organism at a given locus are identical, that is, that they have the same nucleotide for nucleotide exchange at the same place in their sequences. By "hybridization" or "hybridizing," as used herein, is meant the formation of A-T 30 and C-G base pairs between the nucleotide sequence of a fragment of a segment of a polynucleotide and a complementary nucleotide sequence of an oligonucleotide. By complementary is meant that at the locus of each A, C, G or T (or U in a ribonucleotide) in the fragment sequence, the oligonucleotide sequenced has a T, G, C or A, respectively. The hybridized fragment/ oligonucleotide is called a "duplex." WO 2007/129219 PCT/IB2007/001717 12 A "hybridization complex", such as in a sandwich assay, means a complex of nucleic acid molecules including at least the target nucleic acid and a sensor probe. It may also include an anchor probe. As used herein, the term "locus" or "loci" refers to the site of a gene on a 5 chromosome. Pairs of genes, known as "alleles" control the hereditary trait produced by a gene locus. Each animal's particular combination of alleles is referred to as its "genotype". Where both alleles are identical the individual is said to be homozygous for the trait controlled by that gene pair; where the alleles are different, the individual is said to be heterozygous for the trait. 10 A "melting temperature" is meant the temperature at which hybridized duplexes dehybridize and return to their single-stranded state. Likewise, hybridization will not occur in the first place between two oligonucleotides, or, herein, an oligonucleotide and a fragment, at temperatures above the melting temperature of the resulting duplex. It is presently advantageous that the difference in melting point temperatures of oligonucleotide-fragment 15 duplexes of this invention be from about 1 *C to about 10*C so as to be readily detectable. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be single-stranded or double-stranded, but 20 advantageously is double-stranded DNA. "DNA" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or'cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., 25 restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules 30 that are present in the natural source of the nucleic acid. A "nucleoside" refers to a base linked to a sugar. The base may be adenine (A), guanine (G) (or its substitute, inosine (I)), cytosine (C), or thymine (T) (or its substitute, uracil (U)). The sugar may be ribose (the sugar of a natural nucleotide in RNA) or 2 deoxyribose (the sugar of a natural nucleotide in DNA). A "nucleotide" refers to a WO 2007/129219 PCT/IB2007/001717 13 nucleoside linked to a single phosphate group. As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a 5 genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides may be chemically synthesized and may be used as primers or probes. Oligonucleotide means any nucleotide of more than 3 bases in length used to facilitate detection or identification of a target nucleic acid, including probes and primers. 10 A "polymerase" is an enzyme that catalyzes the sequential addition of monomeric units to a polymeric chain, or links two or more monomeric units to initiate a polymeric chain. The "polymerase" will work by adding monomeric units whose identity is determined by and which is complementary to a template molecule of a specific sequence. For example, DNA polymerases such as DNA pol 1 and Taq polymerase add deoxyribonucleotides to the 15 3' end of a polynucleotide chain in a template-dependent manner, thereby synthesizing a nucleic acid that is complementary to the template molecule. Polymerases may be used either to extend a primer once or repetitively or to amplify a polynucleotide by repetitive priming of two complementary strands using two primers. A "thermostable polymerase" refers to a DNA or RNA polymerase enzyme that can withstand extremely high temperatures, 20 such as those approaching 100"C. Often, thermostable polymerases are derived from organisms that live in extreme temperatures, such as Thermus aquaticus. Examples of thermostable polymerases include Taq, Tth, Pfu, Vent, deep vent, UlTma, and variations and derivatives thereof. A "polynucleotide" refers to a linear chain of.nucleotides connected by a 25 phosphodiester linkage between the 3'-hydroxyl group of one nucleoside and the 5'-hydroxyl group of a second nucleoside which in turn is linked through its 3'-hydroxyl group to the 5' hydroxyl group of a third nucleoside and so on to form a polymer comprised of nucleosides liked by a phosphodiester backbone. A "modified polynucleotide" refers to a polynucleotide in which one or more natural nucleotides have been partially or substantially completely 30 replaced with modified nucleotides. A "primer" is an oligonucleotide, the sequence of at least of portion of which is complementary to a segment of a template DAN which to be amplified or replicated. Typically primers are used in performing the polymerase chain reaction (PCR). A primer hybridized with (or "anneals" to) the template DNA and is used by the polymerase enzyme WO 2007/129219 PCT/IB2007/001717 14 uses as the starting point for the replication/amplification process. The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence 5 of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis 10 of the extension product. "Probes" refer to oligonucleotides nucleic acid sequences of variable length, used in the detection of identical, similar, or complementary nucleic acid sequences by hybridization. An oligonucleotide sequence used as a detection probe may be labeled with a detectable moiety. 15 The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and 20 linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling 25 components, other polynucleotides or solid support. An "isolated" polynucleotide or polypeptide is one that is substantially pure of the materials with which it is associated in its native environment. By substantially free, is meant at least 50%, at least 55%, at least 60%, at least 65%, at advantageously at least 70%, at least 75%, more advantageously at least 80%, at least 85%, even more advantageously at least 30 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, most advantageously at least 98%, at least 99%, at least 99.5%, at least 99.9% free of these materials. An "isolated" nucleic acid molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid WO 2007/129219 PCT/IB2007/001717 15 molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith. The term "polynucleotide encoding a protein" as used herein refers to a DNA 5 fragment or isolated DNA molecule encoding a protein, or the complementary strand thereto; but, RNA is not excluded, as it is understood in the art that thymidine (T) in a DNA sequence is considered equal to uracil (U) in an RNA sequence. Thus, RNA sequences for use in the invention, e.g., for use in RNA vectors, can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences. 10 A DNA "coding sequence" or a "nucleotide sequence encoding" a particular protein, is a DNA sequence which is transcribed and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory elements. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, 15 prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence. "Homology" refers to the percent identity between two polynucleotide or two polypeptide moieties. Two DNA, or two polypeptide sequences are "substantially 20 homologous" to each other when the sequences exhibit at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, preferably at least about 90%, 91%, 92%, 93%, 94% and most preferably at least about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity (100% sequence identity) to the specified DNA or 25 polypeptide sequence. Homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single stranded-specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization 30 experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al. supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra. Two nucleic acid fragments are considered to be "selectively hybridizable" to a polynucleotide if they are capable of specifically hybridizing to a nucleic acid or a variant WO 2007/129219 PCT/IB2007/001717 16 thereof or specifically priming a polymerase chain reaction: (i) under typical hybridization and wash conditions, as described, for example, in Sambrook et al. supra and Nucleic Acid Hybridization, supra, (ii) using reduced stringency wash conditions that allow at most about 25-30% basepair mismatches, for example: 2x SSC, 0.1% SDS, room temperature twice, 30 5 minutes each; then 2x SSC, 0.1% SDS, 37*C once, 30 minutes; then 2 x SSC room temperature twice, 10 minutes each, or (iii) selecting primers for use in typical polymerase chain reactions (PCR) under standard conditions (described for example, in Saiki, et al. (1988) Science 239:487-491). The term "capable of hybridizing under stringent conditions" as used herein refers to 10 annealing a first nucleic acid to a second nucleic acid under stringent conditions as defined below. Stringent hybridization conditions typically permit the hybridization of nucleic acid molecules having at least 70% nucleic acid sequence identity with the nucleic acid molecule being used as a probe in the hybridization reaction. For example, the first nucleic acid may be a test sample or probe, and the second nucleic acid may be the sense or antisense strand of 15 a nucleic acid or a fragment thereof. Hybridization of the first and second nucleic acids may be conducted under stringent conditions, e.g., high temperature and/or low salt content that tend to disfavor hybridization of dissimilar nucleotide sequences. Alternatively, hybridization of the first and second nucleic acid may be conducted under reduced stringency conditions, e.g. low temperature and/or high salt content that tend to favor hybridization of 20 dissimilar nucleotide sequences. Low stringency hybridization conditions may be followed by high stringency conditions or intermediate medium stringency conditions to increase the selectivity of the binding of the first and second nucleic acids. The hybridization conditions may further include reagents such as, but not limited to, dimethyl sulfoxide (DMSO) or formamide to disfavor still further the hybridization of dissimilar nucleotide sequences. A 25 suitable hybridization protocol may, for example, involve hybridization in 6 x SSC (wherein I x SSC comprises 0.015 M sodium citrate and 0.15 M sodium chloride), at 65* Celsius in an aqueous solution, followed by washing with I x SSC at 65* C. Formulae to calculate appropriate hybridization and wash conditions to achieve hybridization permitting 30% or less mismatch between two nucleic acid molecules are disclosed, for example, in Meinkoth et 30 al. (1984) Anal. Biochem. 138: 267-284; the content of which is herein incorporated by reference in its entirety. Protocols for hybridization techniques are well known to those of skill in the art and standard molecular biology manuals may be consulted to select a suitable hybridization protocol without undue experimentation. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, the WO 2007/129219 PCT/IB2007/001717 17 contents of which are herein incorporated by reference in their entirety. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M sodium ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) from about pH 7.0. to about pH 8.3 and the temperature is at least about 300 Celsius for 5 short probes (e.g., 10 to 50 nucleotides) and at least about 60* C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37* Celsius, and a wash in 1-2 x SSC at 50 to 550 Celsius. Exemplary 10 moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCI, 1% SDS at 370 Celsius, and a wash in 0.5-1 x SSC at 55 to 600 Celsius. Exemplary high stringency conditions include hybridization in 50% formamide, I M NaCl, 1% SDS at 37* Celsius, and a wash in 0.1 x SSC at 60 to 650 Celsius. Methods and materials of the invention may be used more generally to evaluate a 15 DNA sample from an animal, genetically type an individual animal, and detect genetic differences in animals. In particular, a sample of genomic DNA from an animal may be evaluated by reference to one or more controls to determine if a SNP, or group of SNPs, in a gene is present. Any method for determining genotype can be used for determining the genotype in the present invention. Such methods include, but are not limited to, amplimer 20 sequencing, DNA sequencing, fluorescence spectroscopy, fluorescence resonance energy transfer (or "FRET")-based hybridization analysis, high throughput screening, mass spectroscopy, microsatellite analysis, nucleic acid hybridization, polymerase chain reaction (PCR), RFLP analysis and size chromatography (e.g., capillary or gel chromatography), all of which are well known to one of skill in the art. In particular, methods for determining 25 nucleotide polymorphisms, particularly single nucleotide polymorphisms, are described in U.S. Patent Nos. 6,514,700; 6,503,710; 6,468,742; 6,448,407; 6,410,231; 6,383,756; 6,358,679; 6,322,980; 6,316,230; and 6,287,766 and reviewed by Chen and Sullivan, Pharmacogenomics J 2003;3(2):77-96, the disclosures of which are incorporated by reference in their entireties. Genotypic data useful in the methods of the invention and methods for the 30 identification and selection of animal traits are based on the presence of SNPs. A "restriction fragment" refers to a fragment of a polynucleotide generated by a restriction endonuclease (an enzyme that cleaves phosphodiester bonds within a polynucleotide chain) that cleaves DNA in response to a recognition site on the DNA. The recognition site (restriction site) consists of a specific sequence of nucleotides typically about WO 2007/129219 PCT/IB2007/001717 18 4-8 nucleotides long. A "single nucleotide polymorphism" or "SNP" refers to a variation in the nucleotide sequence of a polynucleotide that differs from another polynucleotide by a single nucleotide difference. For example, without limitation, exchanging one A for one C, G or T in the entire 5 sequence of polynucleotide constitutes a SNP. It is possible to have more than one SNP in a particular polynucleotide. For example, at one position in a polynucleotide, a C may be exchanged for a T, at another position a G may be exchanged for an A and so on. When referring to SNPs, the polynucleotide is most often DNA. As used herein, a "template" refers to a target polynucleotide strand, for example, 10 without limitation, an unmodified naturally-occurring DNA strand, which a polymerase uses as a means of recognizing which nucleotide it should next incorporate into a growing strand to polymerize the complement of the naturally-occurring strand. Such a DNA strand may be single-stranded or it may be part of a double-stranded DNA template. In applications of the present invention requiring repeated cycles of polymerization, e.g., the polymerase chain 15 reaction (PCR), the template strand itself may become modified by incorporation of modified nucleotides, yet still serve as a template for a polymerase to synthesize additional polynucleotides. A "thermocyclic reaction" is a multi-step reaction wherein at least two steps are accomplished by changing the temperature of the reaction. 20 A "variance" is a difference in the nucleotide sequence among related polynucleotides. The difference may be the deletion of one or more nucleotides from the sequence of one polynucleotide compared to the sequence of a related polynucleotide, the addition of one or more nucleotides or the substitution of one nucleotide for another. The terms "mutation," "polymorphism" and "variance" are used interchangeably herein. As used 25 herein, the term "variance" in the singular is to be construed to include multiple variances; i.e., two or more nucleotide additions, deletions and/or substitutions in the same polynucleotide. A "point mutation" refers to a single substitution of one nucleotide for another. As used herein, the terms "traits", "quality traits" or "physical characteristics" or 30 "phenotypes" refer to advantageous properties of the animal resulting from genetics. Quality traits include, but are not limited to, the animal's genetic ability to efficiently metabolize energy, produce meat or milk, put on intramuscular fat. Physical characteristics include, but are not limited to, marbled, tender or lean meats. The terms may be used interchangeably. A "computer system" refers to the hardware means, software means and data storage WO 2007/129219 PCT/IB2007/001717 19 means used to compile the data of the present invention. The minimum hardware means of computer-based systems of the invention may comprise a central processing unit (CPU), input means, output means, and data storage means. Desirably, a monitor is provided to visualize structure data. The data storage means may be RAM or other means for accessing S computer readable media of the invention. Examples of such systems are microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems running Unix based, Linux, Windows NT, XP or IBM OS/2 operating systems. "Computer readable media" refers to any media which can be read and accessed directly by a computer, and includes, but is not limited to: magnetic storage media such as 10 floppy discs, hard storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories, such as magnetic/optical media. By providing such computer readable media, the data compiled on a particular animal can be routinely accessed by a user, e.g., a feedlot operator. 15 The term "data analysis module" is defined herein to include any person or machine, individually or working together, which analyzes the sample and determines the genetic information contained therein. The term may include a person or machine within a laboratory setting. As used herein, the term "data collection module" refers to any person, object or 20 system obtaining a tissue sample from an animal or embryo. By example and without limitation, the term may define, individually or collectively, the person or machine in physical contact with the animal as the sample is taken, the containers holding the tissue samples, the packaging used for transporting the samples, and the like. Advantageously, the data collector is a person. More advantageously, the data collector is a livestock farmer, a 25 breeder-or a veterinarian The term "network interface" is defined herein to include any person or computer system capable of accessing data, depositing data, combining data, analyzing data, searching data, transmitting data or storing data. The term is broadly defined to be a person analyzing the data, the electronic hardware and software systems used in the analysis, the databases 30 storing the data analysis, and any storage media capable of storing the data. Non-limiting examples of network interfaces include people, automated laboratory equipment, computers and computer networks, data storage devices such as, but not limited to, disks, hard drives or memory chips. The term "breeding history" as used herein refers to a record of the life of an animal WO 2007/129219 PCT/IB2007/001717 20 or group of animals including, but not limited to, the location, breed, period of housing, as well as a genetic history of the animals, including parentage and descent therefrom, genotype, phenotype, transgenic history if relevant and the like. The term "husbandry conditions" as used herein refers to parameters relating to the 5 maintenance of animals including, but not limited to, shed or housing temperature, weekly mortality of a herd, water consumption, feed consumption, ventilation rate and quality, litter condition and the like. The term "veterinary history" as used herein refers to vaccination data of an animal or group of animals, including, but not limited to, vaccine type(s), vaccine batch serial 10 number(s), administered dose, target antigen, method of administering of the vaccine to the recipient animal(s), number of vaccinated animals, age of the animals and the vaccinator. Data relating to a serological or immunological response induced by the vaccine may also be included. "Veterinary history" as used herein is also intended to include the medication histories of the target animal(s) including, but not limited to drug and/or antibiotics 15 administered to the animals including type of administered medication, quantity and dose rates, by whom and when administered, by what route, e.g., oral, subcutaneously and the like, and the response to the medication including desired and undesirable effects thereof The term "diagnostic data" as used herein refers to data relating to the health of the animal(s) other than data detailing the vaccination or medication history of the animal(s). For 20 example, the diagnostic data may be a record of the infections experienced by the animal(s) and the response thereof to medications provided to treat such medications. Serological data including antibody or protein composition of the serum or other biofluids may also be diagnostic data useful to input in the methods of the invention. Surgical data pertaining to the animal(s) may be included, such as the type of surgical manipulation, outcome of the surgery 25 and complications arising from the surgical procedure. "Diagnostic data" may also include measurements of such parameters as weight, morbidity, and other characteristics noted by a veterinary service such as the condition of the skin, feet etc. The term "welfare data" as used herein refers to the collective accumulation of data pertaining to an animal or group of animals including, but not limited to, a breeding history, a 30 veterinary history, a welfare profile, diagnostic data, quality control data, or any combination thereof.. The term "welfare profile" as used herein refers to parameters such as weight, meat density, crowding levels in breeding or rearing enclosures, psychological behavior of the animal, growth rate and quality and the like.
WO 2007/129219 PCT/IB2007/001717 21 The term "quality control" as used herein refers to the desired characteristics of the animal(s). For non-poultry animals such as cattle and sheep for example, such parameters include muscle quantity and density, fat content, meat tenderness, milk yield and quality, breeding ability, and the like. 5 The term "performance parameters" as used herein refers to such factors as meat yield, breeding yield, dairy form, meat quality and yield, productive life and the like that may be the desired goals from the breeding and rearing of the animal(s). Performance parameters may be either generated from the animals themselves, or those parameters desired by a customer or the market. 10 The term "nutritional data" as used herein refers to the composition, quantity and frequency of delivery of feed, including water, provided to the animal(s). The term "food safety" as used herein refers to the quality and quantity of the meat from a livestock animal, including, but not limited to, preparation time, place and manner, storage of the food product, transportation route, inspection records, texture, color, taste, 15 odor, bacterial content, parasitic content and the like. It will be apparent to those of skill in the art that the data relating to the health and maintenance of the animals may be variously grouped depending upon the source or intention of the data collector and any one grouping herein is not therefore intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same 20 meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. In an embodiment wherein the gene of interest is bovine growth hormone receptor 25 (GHR), the bovine GHR nucleotide sequence can be selected from, but is not limited to, the sequence corresponding to GenBank Accession No. AY643 807 or a fragment thereof or a region of the bovine genome that comprises this sequence. The present invention, therefore, provides isolated nucleic acids that may specifically hybridize to the nucleotide sequence corresponding to GenBank Accession No. AY643807, 30 or the complement thereof, and which comprises the polymorphic site corresponding to nucleotide position 300 in intron 4 of the bovine GHR gene, in particular a specific adenine (A) to guanine (G) mutation at that position. The SNP advantageous in the present invention is associated with certain economically valuable heritable traits relating to meat quality in bovines. Therefore, it is an WO 2007/129219 PCT/IB2007/001717 22 object of the present invention to determine the genotype of a given animal of interest as defined by the GHR locus SNP according to the present invention. It is also contemplated that the genotype of the animal(s) may be defined by additional SNPs within the GHR gene or within other genes identified with desirable traits or other characteristics, and in particular 5 by a panel or panels of SNPs. There are many methods known in the art for determining the sequence of DNA in a sample, and for identifying whether a given DNA sample contains a particular SNP. Any such technique known in the art may be used in performance of the methods of the present invention. 10 The methods of the present invention allow animals with certain economically valuable heritable traits relating to growth, feed intake, efficiency and carcass merit, to be identified based on the presence of SNPs in their genomes and particularly with an SNP located within intron 4 of the GHR gene. The methods further allow, by computer-assisted methods of the invention, to correlate the SNP-associated traits with other data pertinent to 15 the well-being and productive capacity of the animals, or group of animals. In an embodiment wherein the gene of interest is ghrelin, the bovine ghrelin nucleotide sequence can be selected from, but is not limited to, the sequence corresponding to the bovine ghrelin nucleotide sequence described in the Examples or a fragment thereof or a region of the bovine genome that comprises this sequence. 20 The present invention, therefore, provides isolated nucleic acids that may specifically hybridize to the nucleotide sequence corresponding to the sequence corresponding to the bovine ghrelin nucleotide sequence described in the Examples, or the complement thereof, and which comprises the polymorphic site corresponding to nucleotide position in intron 3 of the bovine ghrelin gene, in particular a specific adenine (A) to guanine (G) mutation at that 25 position (see below for sequence data). The SNP advantageous in the present invention is associated with certain economically valuable heritable traits relating to meat quality in bovines. Therefore, it is an object of the present invention to determine the genotype of a given animal of interest as defined by the ghrelin locus SNP according to the present invention. It is also contemplated 30 that the genotype of the animal(s) may be defined by additional SNPs within the ghrelin gene or within other genes identified with desirable traits or other characteristics, and in particular by a panel or panels of SNPs. There are many methods known in the art for determining the sequence of DNA in a sample, and for identifying whether a given DNA sample contains a particular SNP. Any WO 2007/129219 PCT/IB2007/001717 23 such technique known in the art may be used in performance of the methods of the present invention. The methods of the present invention allow animals with certain economically valuable heritable traits relating to growth, feed intake, efficiency and carcass merit, to be 5 identified based on the presence of SNPs in their genomes and particularly with an SNP located within intron 3 of the ghrelin gene. The methods further allow, by computer-assisted methods of the invention, to correlate the SNP-associated traits with other data pertinent to the well-being and productive capacity of the animals, or group of animals. In an embodiment wherein the gene of interest is leptin, the leptin nucleotide 10 sequence can be selected from, but is not limited to, the sequence corresponding to GenBank Accession No. AB070368 (see, e.g., Taniguchi et al., IUBMB Life. 2002 Feb;53(2):131-5) or a fragment thereof or a region of the bovine genome that comprises this sequence, or the sequence corresponding to GenBank Accession No. BTA512639 (see, e.g., Liefers et al., Mamm. Genome 14 (9), 657-663 (2003)) or a fragment thereof or a region of the bovine 15 genome that comprises this sequence The present invention, therefore, provides isolated nucleic acids that may specifically hybridize to the nucleotide sequence corresponding to GenBank Accession No. AB070368, or the complement thereof, and which comprises the polymorphic site corresponding to nucleotide position 528 of the bovine leptin gene, in particular a specific cytosine (C) to 20 thymine (T) mutation at that position. The present invention also provides isolated nucleic acids that may specifically hybridize to the nucleotide sequence corresponding to GenBank Accession No. BTA512639, or the complement thereof, and which comprises the polymorphic site corresponding to nucleotide position 321 of the bovine leptin gene, in particular a specific cytosine (C) to thymine (T) mutation at that position. 25 The SNP advantageous in the present invention is associated with certain economically valuable heritable traits relating to meat quality in bovines. Therefore, it is an object of the present invention to determine the genotype of a given animal of interest as defined by the leptin locus SNP according to the present invention. It is also contemplated that the genotype of the animal(s) may be defined by additional SNPs within the leptin gene 30 or within other genes identified with desirable traits or other characteristics, and in particular by a panel or panels of SNPs. There are many methods known in the art for determining the sequence of DNA in a sample, and for identifying whether a given DNA sample contains a particular SNP. Any such technique known in the art may be used in performance of the methods of the present WO 2007/129219 PCT/IB2007/001717 24 invention. The methods of the present invention allow animals with certain economically valuable heritable traits relating to growth, feed intake, efficiency and carcass merit, to be identified based on the presence of SNPs in their genomes and particularly with an SNP 5 located within the leptin gene. The methods further allow, by computer-assisted methods of the invention, to correlate the SNP-associated traits with other data pertinent to the well-being and productive capacity of the animals, or group of animals. In an embodiment wherein the gene of interest is bovine neuropeptide Y (NPY), the bovine NPY nucleotide sequence can be selected from, but is not limited to, the sequence 10 corresponding to GenBank Accession No. AY491054 (see, e.g., Thue & Buchanan, Anim Genet. 2004 Jun;35(3):245-6) or a fragment thereof or a region of the bovine genome that comprises this sequence. The present invention, therefore, provides isolated nucleic acids that may specifically hybridize to the nucleotide sequence corresponding to GenBank Accession No. AY491054, 15 or the complement thereof, and which comprises the polymorphic site corresponding to nucleotide position 666 in intron 2 of the bovine NPY gene, in particular a specific adenine (A) to guanine (G) mutation at that position. The SNP advantageous in the present invention is associated with certain economically valuable heritable traits relating to meat quality in bovines. Therefore, it is an 20 object of the present invention to determine the genotype of a given animal of interest as defined by the NPY locus SNP according to the present invention. It is also contemplated that the genotype of the animal(s) may be defined by additional SNPs within the NPY gene or within other genes identified with desirable traits or other characteristics, and in particular by a panel or panels of SNPs. 25 There are many methods known in the art for determining the sequence of DNA in a sample, and for identifying whether a given DNA sample contains a particular SNP. Any such technique known in the art may be used in performance of the methods of the present invention. The methods of the present invention allow animals with certain economically 30 valuable heritable traits relating to growth, feed intake, efficiency and carcass merit, to be identified based on the presence of SNPs in their genomes and particularly with an SNP located within intron 2 of the NPY gene. The methods further allow, by computer-assisted methods of the invention, to correlate the SNP-associated traits with other data pertinent to the well-being and productive capacity of the animals, or group of animals.
WO 2007/129219 PCT/IB2007/001717 25 In an embodiment wherein the gene of interest is bovine uncoupling protein 2 (UCP2) gene, the bovine UCP2 nucleotide sequence can be selected from, but is not limited to, the sequence corresponding to GenBank Accession No. XM_614452 or a fragment thereof or a region of the bovine genome that comprises this sequence. 5 The present invention, therefore, provides isolated nucleic acids that may specifically hybridize to the nucleotide sequence corresponding to GenBank Accession No. XM_614452, or the complement thereof, and which comprises the polymorphic site corresponding to nucleotide position 812 of exon 4 in the bovine UCP2 gene, in particular a specific adenine (A) to guanine (G) mutation at that position (UCP2 SNP2). The present invention also 10 provides isolated nucleic acids that may specifically hybridize to the nucleotide sequence (see nucleotide sequence provided below for UCP2 SNP I) which comprises a cytosine (C) to guanine (G) substitution at position 213 in intron 2 of the bovine'UCP2 gene sequence provided (please see nucleotide sequence below). The SNP advantageous in the present invention is associated with certain 15 economically valuable heritable traits relating to meat quality in bovines. Therefore, it is an object of the present invention to determine the genotype of a given animal of interest as defined by the UCP2 locus SNP according to the present invention. It is also contemplated that the genotype of the animal(s) may be defined by additional SNPs within the UCP2 gene or within other genes identified with desirable traits or other characteristics, and in particular 20 by a panel or panels of SNPs. There are many methods known in the art for determining the sequence of DNA in a sample, and for identifying whether a given DNA sample contains a particular SNP. Any such technique known in the art may be used in performance of the methods of the present invention. 25 The methods of the present invention allow animals with certain economically valuable heritable traits relating to growth, feed intake, efficiency and carcass merit, to be identified based on the presence of SNPs in their genomes and particularly with an SNP located within exon 4 or intron 2 of the UCP2 gene. The methods further allow, by computer-assisted methods of the invention, to correlate the SNP-associated traits with other 30 data pertinent to the well-being and productive capacity of the animals, or group of animals. To determine the genotype of a given animal according to the methods of the present invention, it is necessary to obtain a sample of genomic DNA from that animal. Typically, that sample of genomic DNA will be obtained from a sample of tissue or cells taken from that animal. A tissue or cell sample may be taken from an animal at any time in the lifetime of an WO 2007/129219 PCT/IB2007/001717 26 animal but before the carcass identity is lost. The tissue sample can comprise hair, including roots, hide, bone, buccal swabs, blood, saliva, milk, semen, embryos, muscle or any internal organs. In the methods of the present invention, the source of the tissue sample, and thus also the source of the test nucleic acid sample, is not critical. For example, the test nucleic acid 5 can be obtained from cells within a body fluid of the animal, or from cells constituting a body tissue of the animal. The particular body fluid from which cells are obtained is also not critical to the present invention. For example, the body fluid may be selected from the group consisting of blood, ascites, pleural fluid and spinal fluid. Furthermore, the particular body tissue from which cells are obtained is also not critical to the present invention. For example, 10 the body tissue may be selected from the group consisting of skin, endometrial, uterine and cervical tissue. Both normal. and tumor tissues can be used. Typically, the tissue sample is marked with an identifying number or other indicia that relates the sample to the individual animal from which the sample was taken. The identity of the sample advantageously remains constant throughout the methods and systems of the 15 invention thereby guaranteeing the integrity and continuity of the sample during extraction and analysis. Alternatively, the indicia may be changed in a regular fashion that ensures that the data, and any other associated data, can be related back to the animal from which the data. was obtained. The amount/size of sample required is known to those skilled in the art and for 20 example, can be determined by the subsequent steps used in the method and system of the invention and the specific methods of analysis used. 'Ideally, the size/volume of the tissue sample retrieved should be as consistent as possible within the type of sample and the species of animal. For example, for cattle, non-limiting examples of sample sizes/methods include non-fatty meat: 0.0002 gm-10.0 gm; hide: 0.0004 gm-10.0 gm; hair roots: at least one and 25 advantageously greater than five; buccal swabs: 15 to 20 seconds of rubbing with modest pressure in the area between outer lip and gum using, for example, a cytology brush; bone: 0.0002 gm-10.0 gm; blood: 30 il to 50 ml. Generally, the tissue sample is placed in a container that is labeled using a numbering system bearing a code corresponding to the animal, for example, to the animal's ear tag. 30 Accordingly, the genotype of a particular animal is easily traceable at all times. The sampling device and/or container may be supplied to the farmer, a slaughterhouse or retailer. The sampling device advantageously takes a consistent and reproducible sample from individual animals while simultaneously avoiding any cross-contamination of tissue. Accordingly, the size and volume of sample tissues derived from individual animals would be WO 2007/129219 PCT/IB2007/001717 27 consistent. DNA can be isolated from the tissue/cells by techniques known to those skilled in the art (see, e.g., U.S. Patent Nos. 6,548,256 and 5,989,431; Hirota et al. (1989) Jinrui Idengaku Zasshi. 34: 217-23 and John et al. (1991) Nucleic Acids Res. 19:408, the disclosures of which 5 are incorporated by reference in their entireties). For example, high molecular weight DNA may be purified from cells or tissue using proteinase K extraction and ethanol precipitation. DNA, however, may be extracted from an animal specimen using any other suitable methods known in the art. In one embodiment, the presence or absence of the SNP of any of the genes of the 10 present invention may be determined by sequencing the region of the genomic DNA sample that spans the polymorphic locus. Many methods of sequencing genomic DNA are known in the art, and any such method can be used, see for example Sambrook et al. (200 1) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press. For example, as described below, a DNA fragment spanning the location of the SNP of interest can be 15 amplified using the polymerase chain reaction. The amplified region of DNA form can then be sequenced using any method known in the art, for example using an automatic nucleic acid sequencer. The detection of a given SNP can then be performed using hybridization of probes and or using PCR-based amplification methods. Such methods are described in more detail below. 20 The methods of the present invention may use oligonucleotides useful as primers to amplify specific nucleic acid sequences of the GHR, ghrelin, leptin, NPY or UCP2 gene, advantageously of the region encompassing a GHR, ghrelin, leptin, NPY or UCP2 SNP. Such fragments should be of sufficient length to enable specific annealing or hybridization to the nucleic acid sample. The sequences typically will be about 8 to about 44 nucleotides in 25 length. Longer sequences, e.g., from about 14 to about 50, may be advantageous for certain embodiments. The design of primers is well known to one of ordinary skill in the art. Inventive nucleic acid molecules include nucleic acid molecules having at least 70% identity. or homology or similarity with a GHR, ghrelin, leptin, NPY or UCP2 gene or probes or primers derived therefrom such as at least 75% identity or homology or similarity, 30 preferably at least 80% identity or homology or similarity, more preferably at least 85% identity or homology or similarity such as at least 90% identity or homology or similarity, more preferably at least 95% identity or homology or similarity such as at least 97% identity or homology or similarity. The nucleotide sequence similarity or homology or identity can be determined using the "Align" program of Myers and Miller, ("Optimal Alignments in WO 2007/129219 PCT/IB2007/001717 28 Linear Space", CABIOS 4, 11-17, 1988) and available at NCBI. Alternatively or additionally, the terms "similarity" or "identity" or "homology", for instance, with respect to a nucleotide sequence, is intended to indicate a quantitative measure of homology between two sequences. The percent sequence similarity can be calculated as (Nrf - Ndy)* 1 OO/Nrgf, 5 wherein Ndg is the total number of non-identical residues in the two sequences when aligned and wherein Nrg is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence similarity of 75% with the sequence AATCAATC (Nrg = 8; Ndy= 2 ). Alternatively or additionally, "similarity" with respect to sequences refers to the number of positions with identical nucleotides divided by the number 10 of nucleotides in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur and Lipman, 1983 PNAS USA 80:726), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially 15 available programs (e.g., Intelligenetics TM Suite, Intelligenetics Inc. CA).. When RNA sequences are said to be similar, or have a degree of sequence identity with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. A probe or primer can be any stretch of at least 8, preferably at least 10, more preferably at least 12, 13, 14, or 15, such as at least 20, e.g., at least 23 or 25, for instance at 20 least 27 or 30 nucleotides in a GHR, ghrelin, leptin, NPY or UCP2 gene which are unique to a GHR, ghrelin, leptin, NPY or UCP2 gene. As to PCR or hybridization primers or probes and optimal lengths therefor, reference is also made to Kajimura et al., GATA 7(4):71-79 (1990). RNA sequences within the scope of the invention are derived from the DNA 25 sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences. The oligonucleotides can be produced by a conventional production process for general oligonucleotides. They can be produced, for example, by a chemical synthesis process or by a microbial process that makes use of a plasmid vector, a phage vector or the 30 like. Further, it is suitable to use a nucleic acid synthesizer. To label an oligonucleotide with the fluorescent dye, one of conventionally known labeling methods can be used (Tyagi & Kramer (1996) Nature Biotechnology 14: 303-308; Schofield et al. (1997) Appl. and Environ. Microbiol. 63: 1143-1147; Proudnikov & WO 2007/129219 PCT/IB2007/001717 29 Mirzabekov (1996) Nucl. Acids Res. 24: 4532-4535). Alternatively, the oligonucleotide may be labeled with a radiolabel e.g., 3 H, 12 5 , 35 S, 1 4 C, 3 2 P, etc. Well-known labeling methods are described, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press. The label is coupled directly or indirectly to a component 5 of the oligonucleotide according to methods well known in the art. Reversed phase chromatography or the like used to provide a nucleic. acid probe for use in the present invention can purify the synthesized oligonucleotide labeled with a marker. An advantageous probe form is one labeled with a fluorescent dye at the 3'- or 5'-end and containing G or C as the base at the labeled end. If the 5'-end is labeled and the 3'-end is not labeled, the OH 10 group on the C atom at the 3'-position of the 3'-end ribose or deoxyribose may be modified with a phosphate group or the like although no limitation is imposed in this respect. During the hybridization of the nucleic acid target with the probes, stringent conditions may be utilized, advantageously along with other stringency affecting conditions, to aid in the hybridization. Detection by differential disruption is particularly advantageous 15 to reduce or eliminate slippage hybridization among probes and target, and to promote more effective hybridization. In yet another aspect, stringency conditions may be varied during the hybridization complex stability determination so as to more accurately or quickly determine whether a SNP is present in the target sequence. One method for determining the genotype at the polymorphic gene locus encompasses 20 obtaining a nucleic acid sample, hybridizing the nucleic acid sample with a probe, and disrupting the hybridization to determine the level of disruption energy required wherein the probe has a different disruption energy for one allele as compared to another allele. In one example, there can be a lower disruption energy, e.g., melting temperature, for an allele that harbors a cytosine residue at a polymorphic locus, and a higher required energy for an allele 25 with a different residue at that polymorphic locus. This can be achieved where the probe has 100% homology with one allele (a perfectly matched probe), but has a single mismatch with the alternative allele. Since the perfectly matched probe is bound more tightly to the target DNA than the mis-matched probe, it requires more energy to cause the hybridized probe to dissociate. 30 In a further step of the above method, a second ("anchor") probe may be used. Generally, the anchor probe is not specific to either allele, but hybridizes regardless of what nucleotide is present at the polymorphic locus. The anchor probe does not affect the disruption energy required to disassociate the hybridization complex but, instead, contains a complementary label for using with the first ("sensor") probe.
WO 2007/129219 PCT/IB2007/001717 30 Hybridization stability may be influenced by numerous factors, including thermoregulation, chemical regulation, as well as electronic stringency control, either alone or.in combination with the other listed factors. Through the use of stringency conditions, in either or both of the target hybridization step or the sensor oligonucleotide stringency step, 5 rapid completion of the process may be achieved. This is desirable to achieve properly indexed hybridization of the target DNA to attain the maximum number of molecules at a test site with an accurate hybridization complex. By way of example, with the use of stringency, the initial hybridization step may be completed in ten minutes or less, more advantageously five minutes or less, and most advantageously two minutes or less. Overall, the analytical 10 process may be completed in less than half an hour. In one mode, the hybridization complex is labeled and the step of determining the amount of hybridization includes detecting the amounts of labeled hybridization complex at the test sites. The detection device and method may include, but is not limited to, optical imaging, electronic imaging, imaging with a CCD camera, integrated optical imaging, and 15 mass spectrometry. Further, the amount of labeled or unlabeled probe bound to the target may be quantified. Such quantification may include statistical analysis. The labeled portion of the complex may be the target, the stabilizer, the probe or the hybridization complex in toto. Labeling may be by fluorescent labeling selected from the group of, but not limited to, Cy3, Cy5, Bodipy Texas Red, Bodipy Far Red, Lucifer Yellow, Bodipy 630/650-X, Bodipy 20 R6G-X and 5-CR 6G. Colormetric labeling, bioluminescent labeling and/or chemiluminescent labeling may further accomplish labeling. Labeling further may include energy transfer between molecules in the hybridization complex by perturbation analysis, quenching, electron transport between donor and acceptor molecules, the latter of which may be facilitated by double stranded match hybridization complexes. Optionally, if the 25 hybridization complex is unlabeled, detection may be accomplished by measurement of conductance differential between double stranded and non-double stranded DNA. Further, direct detection may be achieved by porous silicon-based optical interferometry or by mass spectrometry. In using mass spectrometry no fluorescent or other label is necessary. Rather detection is obtained by extremely high levels of mass resolution achieved by direct 30 measurement, for example, by time of flight (TOF) or by electron spray ionization (ESI). Where mass spectrometry is contemplated, probes having a nucleic acid sequence of 50 bases or less are advantageous. The label may be amplified, and may include, for example, branched or dendritic DNA. If the target DNA is purified, it may be un-amplified or amplified. Further, if the WO 2007/129219 PCT/IB2007/001717 31 purified target is amplified and the amplification is an exponential method, it may be, for example, PCR amplified DNA or strand displacement amplification (SDA) amplified DNA. Linear methods of DNA amplification such as rolling circle or transcriptional runoff may also be used. 5 Where it is desired to amplify a fragment of DNA that comprises an SNP according to the present invention, the forward and reverse primers may have contiguous stretches of about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 9, 20, 2 1, 2 2 , 2 3 , 2 4 , 2 5, 2 6 , 27, 28, 29, 30 or any other length up to and including about 50 nucleotides in length. The sequences to which the forward and reverse primers anneal are advantageously located on either side of the 10 particular nucleotide position that is substituted in the SNP to be amplified. A detectable label can be incorporated into a nucleic acid during at least one cycle of an amplification reaction. Spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means can detect such labels. Useful labels in the present invention include fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, 15 and the like), radiolabels (e.g., 3 H, 1251, 35S, "C, 32 P, etc.), enzymes (e.g. horseradish peroxidase, alkaline phosphatase etc.) colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads. The label is coupled directly or indirectly to a component of the assay according to methods well known in the art. As indicated above, a wide variety of labels are used, with the choice of label depending on 20 sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions. Non-radioactive labels are often attached by indirect means. Polymerases can also incorporate fluorescent nucleotides during synthesis of nucleic acids. Reagents allowing the sequencing of reaction products can be utilized herein. For 25 example, chain-terminating nucleotides will often be incorporated into a reaction product during one or more cycles of a reaction. Commercial kits containing the reagents most typically used for these methods of DNA sequencing are available and widely used. PCR exonuclease digestion methods for DNA sequencing can also be used. Many methods of sequencing genomic DNA are known in the art, and any such method can be used, see for 30 example Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press. For example, as described below, a DNA fragment spanning the location of the SNP of interest can amplified using the polymerase chain reaction or some other cyclic polymerase mediated amplification reaction. The amplified region of DNA can then be sequenced using any method known in the art. Advantageously, the nucleic acid WO 2007/129219 PCT/IB2007/001717 32 sequencing is by automated methods (reviewed by Meldrum, (2000) Genome Res. 10: 1288 303, the disclosure of which is incorporated by reference in its entirety), for example using a Beckman CEQ 8000 Genetic Analysis System (Beckman Coulter Instruments, Inc.). Methods for sequencing nucleic acids include, but are not limited to, automated fluorescent 5 DNA sequencing (see, e.g., Watts & MacBeath, (2001) Methods Mol Biol. 167: 153-70 and MacBeath et al. (2001) Methods Mol Biol. 167:119-52), capillary electrophoresis (see, e.g., Bosserhoff et al. (2000) Comb Chem High Throughput Screen. 3: 455-66), DNA sequencing chips (see, e.g., Jain, (2000) Pharmacogenomics. 1: 289-307), mass spectrometry (see, e.g., Yates, (2000) Trends Genet. 16: 5-8), pyrosequencing (see, e.g., Ronaghi, (2001) Genome 10 Res. 11: 3-11), and ultrathin-layer gel electrophoresis (see, e.g., Guttman & Ronai, (2000) Electrophoresis. 21: 3952-64), the disclosures of which are hereby incorporated by reference in their entireties. The sequencing can also be done by a commercial company. Examples of such companies include, but are not limited to, the University of Georgia Molecular Genetics Instrumentation Facility (Athens, Georgia) or SeqWright DNA Technologies Services 15 (Houston, Texas). An SNP-specific probe can also be used in the detection of the SNP in amplified specific nucleic acid sequences of the target gene, such as the amplified PCR products generated using the primers described above. In certain embodiments, these SNP-specific probes consist of oligonucleotide fragments. Advantageously, the fragments are of sufficient 20 length to provide specific hybridization to the nucleic acid sample. The use of a hybridization probe of between 10 and 50 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 12 bases in length are generally advantageous, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree 25 of particular hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having stretches of 16 to 24 nucleotides, or even longer where desired. A tag nucleotide region may be included, as at the 5' end of the primer that may provide a site to which an oligonucleotide sequencing primer may hybridize to facilitate the sequencing of multiple PCR samples. 30 The probe sequence must span the particular nucleotide position that may be substituted in the particular SNP to be detected. Advantageously, two or more different "allele-specific probes" may be used for analysis of a SNP, a first allele-specific probe for detection of one allele, and a second allele-specific probe for the detection of the alternative allele.
WO 2007/129219 PCT/IB2007/001717 33 It will be understood that this invention is not limited to the particular primers and probes disclosed herein and is intended to encompass at least'nucleic acid sequences that are hybridizable to the nucleotide sequence disclosed herein, the complement or a fragment thereof, or are functional sequence analogs of these sequences. It is also contemplated that a 5 particular trait of an animal may be determined by using a panel of SNPs associated with that trait. Several economically relevant traits may be characterized by the presence or absence of one or more SNPs and by a plurality of SNPs in different genes. One or more panels of SNPs may be used in the methods of the invention to define the phenotypic profile of the subject animal. 10 Homologs (i.e., nucleic acids derived from other species) or other related sequences (e.g., paralogs) can be obtained under conditions of standard or stringent hybridization conditions with all or a portion of the particular sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning. The genetic markers, probes thereof, methods, and kits of the invention are also useful 15 in a breeding program to select for breeding those animals having desirable phenotypes for various economically important traits, such as improved meat quality and yield, in particular meat tenderness. Continuous selection and breeding of animals, such as livestock, that are at least heterozygous and advantageously homozygous for desirable alleles of the GHR, ghrelin, leptin, NPY and UCP2 gene polymorphic sites associated with economically relevant traits of 20 growth, feed intake, efficiency and carcass merit, would lead to a breed, line, or population having higher numbers of offspring with economically relevant traits of growth, feed intake, efficiency and carcass merit. Thus, the GHR, ghrelin, leptin, NPY and UCP2 SNPs of the present invention can be used as a selection tool. One aspect of the present invention provides for grouping animals and methods for 25 managing livestock production comprising grouping livestock animals such as cattle according the genotype as defined by panels of SNPs, each panel comprising at least one SNP, one or more of which are GHR, ghrelin, leptin, NPY and UCP2 SNPs of the present invention. Other SNPs that may be included in panels of SNPs include, but not limited to, calpastatin, UASMS1, UASMS2, UASMS3 and/or EXON2-FB SNPs of the ob loci defining 30 the same phenotypic character. The genetic selection and grouping methods of the present invention can be used in conjunction with other conventional phenotypic grouping methods such as grouping animals by visible characteristics such as weight, frame size, breed traits, and the like. The methods of the present invention provide for producing cattle having improved heritable traits, and can be used to optimize the performance of livestock herds in WO 2007/129219 PCT/IB2007/001717 34 areas such as breeding, food consumption, carcass/meat quality and milk production. The present invention provides methods of screening livestock to determine those more likely to develop a desired body condition by identifying the presence or absence of one or more of which are GHR, ghrelin, leptin, NPY and UCP2 polymorphism in genes that is correlated 5 with that meat quality. As described above, and in the Examples, there are various phenotypic traits with which the SNPs of the present invention may be associated. Each of the phenotypic and genetic traits can be tested using the methods described in the Examples, or using any suitable methods known in the art. Using the methods of the invention, a farmer, or feed lot 10 operator, or the like, can group cattle according to each animal's genetic propensity for a desired trait such as growth rate, feed intake or feeding behavior, as determined by SNP genotype. The cattle are tested to determine homozygosity or heterozygosity with respect to the SNP alleles of one or more genes so that they can be grouped such that each pen contains cattle with like genotypes. Each pen of animals is then fed and otherwise maintained in a 15 manner and for a time determined by the feed lot operator, and then slaughtered. Thus, a feeder is presented with opportunities for considerable efficiencies. At present, for example, the feeder may feed his cattle in the same manner, incurring the same costs for each animal, and typically, with excellent management practices, perhaps 40% will grade AAA and receive the premium price for the palatability grade depending on several 20 other factors, such as age of animal, since cattle between 17-24 months of age have increased marbling compared to their younger counterparts. Approximately 55% of cattle are slaughtered at an age under 16 months, and 45% would be slaughtered at an age over 17 months. Of these, a significant number will have excess fat and will thus receive a reduced yield grade. The balance of the cattle, 60%, will grade less than AAA, and thus receive a 25 reduced price, although the feedlot costs incurred by the operator will be the same. Grouping and feeding the cattle by genotype, as well as by other factors such as the overall welfare profile, which includes husbandry and veterinary data, allows the feeder to treat each group differently with a view to increasing profit by maximizing, for example, the number of cattle providing marketable tender meat. 30 The individual genotypic data derived from a panel or panels of SNPs of each animal or a herd or flock of animals can be recorded and associated with various other data of the animal, e.g. health information, parentage, husbandry conditions, vaccination history, herd or flock records, subsequent food safety data and the like. Such information can be forwarded to a government agency to provide traceability of an animal or meat product, or it may serve WO 2007/129219 PCT/IB2007/001717 35 as the basis for breeding, feeding and marketing information. Once the data has or has not been associated with other data, the data is stored in an accessible database, such as, but not limited to, a computer database or a microchip implanted in the animal. The methods of the invention may provide an analysis of the input data that may be compared with parameters 5 desired by the operator. These parameters include, but are not limited to, such as breeding goals, egg laying targets, vaccination levels of a flock or herd. If the performance or properties of the animals deviates from the desired goals, the computer-based methods may trigger an alert to allow the operator to adjust vaccination doses, medications, feed etc accordingly. 10 The results of the analysis provide data that is associated with the individual animal or to the herd in whole or in part from which the sample was taken. The data is then kept in an accessible database, and may or may not be associated with other data from that particular individual or from other animals. Data obtained from individual animals may be stored in a database that can be 15 integrated or associated with and/or cross-matched to other databases. The database along with the associated data allows information about the individual animal to be known through every stage of the animal's life, i.e., from conception to consumption of the animal product. The accumulated data and the combination of the genetic data with other types of data of the animal provides access to information about parentage, identification of herd or flock, 20 health information including vaccinations, exposure to diseases, feed lot location, diet and ownership changes. Information such as dates and results of diagnostic or routine tests are easily stored and attainable. Such information would be especially valuable to companies, particularly those who seek superior breeding lines. Each animal may be provided with a unique identifier. The animal can be tagged, as 25 in traditional tracing programs or have implant computer chips providing stored and readable data or provided with any other identification method which associates the animal with its unique identifier. The database containing the SNP-based genotype results for each animal or the data for each animal can be associated or linked to other databases containing data; for example, 30 which may be helpful in selecting traits for grouping or sub-grouping of an animal. For example, and not for limitation, data pertaining to animals having particular vaccination or medication protocols, can optionally be further linked with data pertaining to animals having food from certain food sources. The ability to refine a group of animals is limited only by the traits sought and the databases containing information related to those traits.
WO 2007/129219 PCT/IB2007/001717 36 Databases that can usefully be associated with the methods of the invention include, but are not limited to, specific or general scientific data. Specific data includes, but is not limited to, breeding lines, sires, dames, and the like, other animals' genotypes, including whether or not other specific animals possess specific genes, including transgenic genetic 5 elements, location of animals which share similar or identical genetic characteristics, and the like. General data includes, but is not limited to, scientific data such as which genes encode for specific quality characteristics, breed association data, feed data, breeding trends, and the like. One method of the present invention includes providing the animal owner or customer 10 with sample collection equipment, such as swabs and vials useful for collecting samples from which genetic data may be obtained. The vials are packaged in a container that is encoded with identifying indicia. Advantageously, the packaging is encoded with a bar code label. The vials are encoded with the same identifying indicia, advantageously with a matching bar code label. Optionally, the packaging contains means for sending the vials to a laboratory for 15 analysis. The optional packaging is also encoded with identifying indicia, advantageously with a bar code label. The method optionally includes a system wherein a database account is established upon ordering the sampling equipment. The database account identifier corresponds to the identifying indicia of the vials and the packaging. Upon shipment of the sampling equipment 20 in fulfillment of the order, the identifying indicia are recorded in a database. Advantageously, the identifier is a bar code label which is scanned when the vials are sent. When the vials are returned to the testing facility, the identifier is again recorded and matched to the information previously recorded in the database upon shipment of the vial to the customer. Once the genotyping is completed, the information is recorded in the database and 25 coded with the unique identifier. Test results are also provided to the customer or animal owner. The data stored in the genotype database can be integrated with or compared to other data or databases for the purpose of identifying animals based on genetic propensities. Other data or databases include, but are not limited to, those containing information related to SNP 30 based DNA testing, vaccination, SUREBRED pre-conditioning program, estrus and pregnancy results, hormone levels, food safety/contamination, somatic cell counts, mastitis occurrence, diagnostic test results, milk protein levels, milk fat, vaccine status, health records, mineral levels, trace mineral levels, herd performance, and the like. The present invention, therefore, encompasses computer-assisted methods for tracking WO 2007/129219 PCT/IB2007/001717 37 the breeding and veterinary histories of livestock animals encompassing using a computer based system comprising a programmed computer comprising a processor, a data storage system, an input device and an output device, and comprising the steps of generating a profile of a livestock animal by inputting into the programmed computer through the input device 5 genotype data of the animal, wherein the genotype may be defined by a panel of at least two single nucleotide polymorphisms that predict at least one physical trait of the animal, inputting into the programmed computer through the input device welfare data of the animal, correlating the inputted welfare data with the phenotypic profile of the animal using the processor and the data storage system, and outputting a profile of the animal or group of 10 animals. to the output device. The databases and the analysis thereof will be accessible to those to whom access has been provided. Access can be provided through rights to access or by subscription to specific portions of the data. For example, the database can be accessed by owners of the animal, the test site, the entity providing the sample to the test site, feedlot personnel, and veterinarians. 15 The data can be provided in any form such as by accessing a website, fax, email, mailed correspondence, automated telephone, or other methods for communication. This data can also be encoded on a portable storage device, such as a microchip, that can be implanted in the animal. Advantageously, information can be read and new information added without removing the microchip from the animal. 20 The present invention comprises systems for performing the methods disclosed herein. Such systems comprise devices, such as computers, internet connections, servers, and storage devices for data. The present invention also provides for a method of transmitting data comprising transmission of information from such methods herein discussed or steps thereof, e.g., via telecommunication, telephone, video conference, mass communication, e.g., 25 presentation such as a computer presentation (e.g. POWERPOINT), internet, email, documentary communication such as computer programs (e.g. WORD) and the like. Systems of the present invention may comprise a data collection module, which includes a data collector to collect data from an animal or embryo and transmit the data to a data analysis module, a network interface for receiving data from the data analysis module, 30 and optionally further adapted to combine multiple data from one or more individual animals, and to transmit the data via a network to other sites, or to a storage device. More particularly, systems of the present invention comprise a data collection module, a data analysis module, a network interface for receiving data from the data analysis module, and optionally further adapted to combine multiple data from one or more individual WO 2007/129219 PCT/IB2007/001717 38 animals, and to transmit the data via a network to other sites, and/or a storage device. For example, the data collected by the data collection module leads to a determination of the absence or presence of a SNP of a gene in the animal or embryo, and for example, such data is transmitted to a feedstock site when the feeding regimen of the animal is planned. 5 In one embodiment where the data is implanted on a microchip on a particular animal, the farmer can optimize the efficiency of managing the herd because the farmer is able to identify the genetic predispositions of an individual animal as well as past, present and future treatments (e.g., vaccinations and veterinarian visits). The invention, therefore also provides for accessing other databases, e.g., herd or flock data relating to genetic tests and data 10 performed by others, by datalinks to other sites. Therefore, data from other databases can be transmitted to the central database of the present invention via a network interface for receiving data from the data analysis module of the other databases. The invention relates to a computer system and a computer readable media for compiling data on an animal, the system containing inputted data on that animal, such as but 15 not limited to, vaccination and medication histories, DNA testing, thyroglobulin testing, leptin, MMI (Meta Morphix Inc.), Bovine spongiform encephalopathy (BSE) diagnosis, brucellosis vaccination, FMD (foot and mouth disease) vaccination, BVD (bovine viral diarrhea) vaccination, SUREBRED pre-conditioning program, estrus and pregnancy results, tuberculosis, hormone levels, food safety/contamination, somatic cell counts, mastitis 20 occurrence, diagnostic test results, milk protein levels, milk fat, vaccine status, health records, mineral levels, trace mineral levels, herd performance, and the like. The data of the animal can also include prior treatments as well as suggested tailored treatment depending on the genetic predisposition of that animal toward a particular disease. The invention also provides for a computer-assisted method for improving animal 25 production comprising using a computer system, e.g., a programmed computer comprising a processor, a data storage system, an input device and an output device, the steps of inputting into the programmed computer through the input device data comprising a breeding, veterinary, medication, diagnostic data and the like of an animal, correlating a physical characteristic predicted by the genotype using the processor and the data storage system, 30 outputting to the output device the physical characteristic correlated to the genotype and feeding the animal a diet based upon the physical characteristic, thereby improving livestock production. The invention further provides for a computer-assisted method for optimizing efficiency of feed lots for livestock comprising using a computer system, e.g., a programmed WO 2007/129219 PCT/IB2007/001717 39 computer comprising a processor, a data storage system, an input device and an output device, and the steps of inputting into the programmed computer through the input device data comprising a breeding, veterinary etc history of an animal, correlating the breeding, veterinary etc histories using the processor and the data storage system, outputting to the 5 output device the physical characteristic correlated to the genotype and feeding the animal a diet based upon the physical characteristic, thereby optimizing efficiency of feed lots for livestock. The invention further comprehends methods of doing business by providing access to such computer readable media and/or computer systems and/or data collected from animals to 10 users; e.g., the media and/or sequence data can be accessible to a user, for instance on a subscription basis, via the Internet or a global communication/computer network; or, the computer system can be available to a user, on a subscription basis. In one embodiment, the invention provides for a computer system for managing livestock comprising physical characteristics and databases corresponding to one or more 15 animals. In another embodiment, the invention provides for computer readable media for managing livestock comprising physical characteristics and veterinary histories corresponding to one or more animals. The invention further provides methods of doing business for managing livestock comprising providing to a user the computer system and media described above or physical characteristics and veterinary histories corresponding to 20 one or more animals. The invention further encompasses methods of transmitting information obtained in any method or step thereof described herein or any information described herein, e.g., via telecommunications, telephone, mass communications, mass media, presentations, internet, email, etc. The invention further encompasses kits useful for screening nucleic acid isolated from 25 one or rhore bovine individuals for allelic variation of any one of the GHR, ghrelin, leptin, NPY and UCP2 genes, and in particular for any of the SNPs described herein, wherein the kits may comprise at least one oligonucleotide selectively hybridizing to a nucleic acid comprising any one of the one or more of which are GHR, ghrelin, leptin, NPY and UCP2 sequences described herein and instructions for using the oligonucleotide to detect variation 30 in the nucleotide corresponding to the SNP of the isolated nucleic acid. One embodiment of this aspect of the invention provides an an oligonucleotide that specifically hybridizes to the isolated nucleic acid molecule of this aspect of the invention, and wherein the oligonucleotide hybridizes to a portion of the isolated nucleic acid molecule comprising any one of the polymorphic sites in the GHR, ghrelin, leptin, NPY and UCP2 WO 2007/129219 PCT/IB2007/001717 40 sequences described herein. Another embodiment of the invention is an oligonucleotide that specifically hybridizes under high stringency conditions to any one of the polymorphic sites of the GHR, ghrelin, leptin, NPY and UCP2 genes, wherein the oligonucleotide is between about 18 5 nucleotides and about 50 nucleotides. In another embodiment of the invention, the oligonucleotide comprises a central nucleotide specifically hybridizing with a GHR, ghrelin, leptin, NPY or UCP2 gene polymorphic site of the portion of the nucleic acid molecule. Another aspect of the invention is a method of identifying a GHR, ghrelin, leptin, 10 NPY or UCP2 polymorphism in a nucleic acid sample comprising isolating a nucleic acid molecule encoding GHR, ghrelin, leptin, NPY or UCP2 or a fragment thereof and determining the nucleotide at the polymorphic site. Another aspect of the invention is a method of screening cattle to determine those bovines more likely to exhibit a biological difference in meat quality comprising the steps of 15 obtaining a sample of genetic material from a bovine; and assaying for the presence of a genotype in the bovine which is associated with meat quality, the genotype characterized by a polymorphism in any one of the GHR, ghrelin, leptin, NPY or UCP2 genes. In other embodiments of this aspect of the invention, the step of assaying is selected from the group consisting of: restriction fragment length polymorphism (RFLP) analysis, 20 minisequencing, MALD-TOF, SINE, heteroduplex analysis, single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE). In various embodiments of the invention, the method may further comprise the step of amplifying a region of the GHR, ghrelin, leptin, NPY or UCP2 gene or a portion thereof that 25 contains the polymorphism. In other embodiments of the invention, the amplification may include the step of selecting a forward and a reverse sequence primer capable of amplifying a region of the GHR, ghrelin, leptin, NPY or UCP2 gene. Another aspect of the invention is a computer-assisted method for predicting which livestock animals possess a biological difference in meat quality comprising: using a 30 computer system, e.g., a programmed computer comprising a processor, a data storage system, an input device and an output device, the steps of: (a) inputting into the programmed computer through the input device data comprising a GHR, ghrelin, leptin, NPY or UCP2 genotype of an animal, (b) correlating a growth, feed intake, efficiency or carcass merit quality predicted by the GHR, ghrelin, leptin, NPY or UCP2 genotype using the processor WO 2007/129219 PCT/IB2007/001717 41 and the data storage system and (c) outputting to the output device the meat quality correlated to the GHR, ghrelin, leptin, NPY or UCP2 genotype, thereby predicting which livestock animals. possess a particular growth, feed intake, efficiency or carcass merit quality. Yet another aspect of the invention is a method of doing business for managing 5 livestock comprising providing to a user computer system for managing livestock comprising physical characteristics and genotypes corresponding to one or more animals or a computer readable media for managing livestock comprising physical characteristics and genotypes corresponding to one or more animals or physical characteristics and genotypes corresponding to one or more animals. 10 The invention will now be further described by way of the following non-limiting examples. EXAMPLES Example 1: Growth Hormone Receptor SNPs This Example illustrates associations between a single nucleotide polymorphism 15 (SNP) in the growth hormone receptor (GHR gene) with economically relevant measures of feed intake, growth, and carcass quality in beef cattle. The SNP is a specific A to G mutation at the 300 nucleotide position in intron 4 of the bovine GHR gene. The GHR gene is bound by the GH gene in a homodimeric constellation resulting in the initiation of signal transduction mechanisms and the subsequent activation of many 20 hormonal systems in the regulation of growth promotion lipid, nitrogen, mineral and carbohydrate metabolism and includes effects on protein synthesis, protein degradation, and regulation of protein and nitrogen retention fat synthesis, increase fatty acid oxidation, stimulate fatty acid mobilization from body adipose tissues. Treatment of animals, especially ruminant livestock, with growth hormone results in decreased feed intake, increased average 25 daily gain, increased feed efficiency, decreased fat accretion and increased protein accretion. The experimental animals used in this study were Continental x British hybrid beef steers sired by Angus, Charolais or University of Alberta Hybrid bulls. Feed intake, growth and carcass data were collected over two years under feedlot conditions at the University of Alberta's Kinsella beef cattle research station. Genomic DNA was extracted from blood 30 samples using a standard high salt phenol/chloroform extraction method. Genotyping of the SNP was carried out using the Illumina GoldenGate assay on the BeadStation system (Illumina Inc., San Diego, CA), which allows the simultaneous genotyping of 1,536 SNPs using 250 ng of genomic DNA per sample. The SNP analyzed is an A to G nucleotide substitution at the 300 nucleotide position WO 2007/129219 PCT/IB2007/001717 42 in intron 4 of the GHR gene (Accession No. AY643807), which has the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1). Associations of the genotypes for each polymorphism with measures of performance and carcass merit were analyzed using General Linear Mixed Model in SAS. The statistical 5 analyses model included fixed effects of SNP genotype, test year (two levels), contemporary test group nested within year (four levels), breed of sire (three levels), linear covariate of age of animal on test, and random effects of sire and dam of animal. Additive and non-additive genetic effects were estimated for traits that were or tended to be significant (P < 0.10) between different SNP genotypes. 10 Table 1. Genotype and allele frequencies of the GHR SNP in the experimental population of beef cattle. Sire breed Number of Animals AA AG GG G allele frequency Angus 127 86 38 3 0.17 Charolais 92 43 43 6 0.30 15 Hybrid 85 51 28 6 0.24 Total 304 181 109 15 0.23 Table 2. Effect of growth hormone receptor genotypes on performance and carcass merit of beef steers GHR SNP genotype P Trait AA AG GG value' Number of animals 180 109 15 Average daily gain, kg/d 1.47 L 0.03 1.41 ) 0.03 1.65 k 0.07 0.004 Final weight, kg 459.42 ± 6.25 450.50 i 6.66 489.30 & 11.96 0.006 Dry matter intake, kg/d 9.36 h 0.16 9.25 0.17 10.30 J 0.33 0.01 Metabolic mid-weight, kg.7s 92.60 * 0.97 91.32 1.03 96.60 : 1.84 0.01 Slaughter weight, kg 523.82 & 7.10 518.75 * 7.57 557.63 13.60 0.02 LM area, cm 2 81.41 : 0.98 80.95 i 1.08 87.18:* 2.31 0.03 Ultrasound LM area, cm 2 77.23 t 0.60 , 76.63 i 0.70 81.42 & 1.81 0.06 Feed conversion ratio, kg DM/kg of gain 6.63 0.08 6.88 = 0.10 6.43 ± 0.26 0.08 Ultrasound marbling score 5.00 ± 0.07 4.97 0.08 5.29 ± 0.17 0.20 a P values from overall F test. 20 Table 3. Additive and non-additive genetic effects of GHR SNP on performance and carcass merit Dominance Trait Additive Effect P value deviation P value Average daily gain, kg/d -0.18 + 0.07 0.02 0.15 = 0.04 0.001 Final weight, kg -29.88 ± 11.26 0.01 23.86 ± 6.64 0.002 Dry matter intake, kg/d -0.94 ± 0.32 0.01 0.58 0.19 0.006 Metabolic mid-weight, kg-7 5 -4.00 h 1.72 0.03 3.28 ± 1.02 0.004 WO 2007/129219 PCT/IB2007/001717 43 Dominance Trait Additive Effect P value deviation P value Slaughter weight, kg -33.81 12.81 0.02 21.97 7.57 0.008 LM area, cm 2 -5.77 ± 2.33 0.03 3.35 t 1.37 0.03 Ultrasound LM area, cm 2 -4.19 ± 1.89 0.04 2.69 1.12 0.03 Feed conversion ratio, kg of DM/kg of gain 0.20 0.27 0.47 -0.35± 0.16 0.04 Ultrasound marbling score -0.29 0.17 0.11 0.17 0.10 0.10 Feeding duration, min/d -5.61 3.44 0.12 3.48 ± 2.02 0.10 Carcass weight, kg -14.15 1 9.06 0.15 8.44 ± 5.31 0.14 Example 2: Leptin SNPs This Example illustrates associations between two single nucleotide polymorphisms (SNPs) in the bovine leptin gene exon 3 and promoter with measures of feed intake, growth and carcass quality in beef cattle. These two SNPs, UASMS2 (C-T mutation at position 528 5 in AB070368) and A59V (C-T mutation at position 321 in BTA512639) in the bovine leptin gene, and their haplotypes, show strong associations with serum leptin concentration and economically relevant traits of growth, feed intake, efficiency and carcass merit in cattle. Leptin is an adipocyte-derived 16-kDa cytokine-like hormone product of the obese gene (Zhang et al., 1994; Ji et al., 1998) that circulates in the serum in the free and bound 10 forms. Leptin's role as a lipostatic signal regulating whole body energy metabolism through interactions with the leptin receptor in the hypothalamus makes it one of the best physiological markers for the regulation of BW, feed intake, energy expenditure, body fatness, milk yield and composition, and overall carcass quality. The experimental animals used in this study were Continental x British hybrid beef 15 steers sired by Angus, Charolais or University of Alberta Hybrid bulls. Feed intake, growth and carcass data were collected over three years under feedlot conditions at the University of Alberta's Kinsella beef cattle research station. Genomic DNA was extracted from blood samples using a standard high salt phenol/chloroform extraction method. Genotyping of the SNP was carried out using the Illumina GoldenGate assay on the BeadStation system 20 (Illumina Inc., San Diego, CA), which allows the simultaneous genotyping of 1,536 SNPs using 250 ng of genomic DNA per sample. Two SNP and their genotype combinations were analyzed in this report. The first mutation, UASMS2, is a C-T substitution located at nucleotide position 528 in the bovine leptin promoter (GenBank accession no. AB070368). The nucleotide sequence is depicted in 25 FIG. 2 (SEQ ID NO: 2). The second mutation, is a C-T substitution at position 321 (GenBank accession no.
WO 2007/129219 PCT/IB2007/001717 44 BTA512639; EMBL Accession no. AJ512639) that results in an alanine (A; GCG) to valine (V; GTG) at amino acid 59 in the p-helix region of the leptin molecule that is conserved between species. The The nucleotide sequence is depicted in FIG. 3 (SEQ ID NO: 3).. Association of the polymorphisms ot their haplotypes with measures of performance 5 and carcass merit were analyzed using a General Linear Mixed Model in SAS. The statistical analyses model included fixed effects of SNP genotype, test year (three levels), contemporary test group nested within year (six levels), breed of sire (three levels), linear covariate of age of animal on test, and random effects of sirie and dam of animal. Results of these analyses are shown in the following tables. Table 4. Association of UASMS2 SNP in the leptin promoter (LS means ± SE) with measures of serum leptin 'concentration, performance, feed efficiency, ultrasound, and carcass merit in composite crossbred cattle (n = 464) UASMS2 SNP genotype Trait CC CT TT Pvalue Number of animals 306 146 12 Serum leptin level, ng/mL 13.04 i 0.38c 13.94 0.54c 19.20 ± 1.53b <0.001 Phenotypic RFI, kg/d -0.07 ± 0.07c 0.16 0.09b 0.13 ± 0.26b 0.09 Genetic RFI, kg/d -0.21 ± 0.07c 0.01 i 0.09b 0.04 i 0.26b 0.10 Feed conversion, kg DM/kg gain 7.21 ± 0.11 7.37 ± 0.14 7.22 ± 0.33 0.47 Dry matter intake, kg/d 10.33 ± 0.13c 10.71 ± 0.17bc 11.09 i 0.42b 0.036 Average daily gain, kg/d 1.47 0.03 1.45 ± 0.03 1.53 ± 0.08 0.52 Metabolic BW, kgO- 75 90.65 i 0.68 90.96 i 0.83 92.58 1.84 0.56 Backfat gain, mm/d (x 10-2) 3.40 ± 0.10c 3.33 ± 0.10e 4.60 i 0.40b 0.017 Marbling gain, units/d (x 10-2) 0.70 ± 0.02c 0.70 ± 0.04c 1.00 ± 0.10b 0.05 Ultrasound backfat, mm 8.93 ± 0.20d 9.09 0.28c 11.38 ± 0.83b 0.017 Ultrasound marbling score 5.07 i 0.06c 5.22 ± 0.08b 5.58 i 0.20b 0.023 Ultrasound LM area, cm 2 83.61 ± 0.56 83.83 ± 0.80 80.73 ± 2.29 0.42 Number of animals 255 118 8 Carcass grade fat, mm 10.32 i 0.30c 10.58 i 0.44c 13.37 ± 1.
3 6b 0.09 Average carcass backfat, mm 11.82 i 0.29e 12.13 ± 0.43c 14.82 ± 1.36b 0.09 LM area 84.18 ± 0.72 83.71 i 0.98 82.86 i 2.75 0.83 Carcass marbling score 2.47 0.04 2.54 i 0.06 2.65 i 0.17 0.35 Lean meat yield 58.17 ± 0.17 57.98 i 0.44 55.88 ± 1.26 0.17 10 aP value from overall F test b, c. d Means in rows followed by different superscripts are different (P < 0.05).
WO 2007/129219 PCT/IB2007/001717 45 Table 5. Association of A59V SNP in leptin exon 3 (LS means ± SE) with measures of serum leptin concentration, performance, feed efficiency, feeding behaviour, and carcass merit in composite crossbred cattle. A59V SNP genotypes Trait CC CT TT P value Number of animals 31 174 259 Serum leptin level, ng/mL 10.80 ± 0.98d 13.40 i 0.401 14.43 ± 0.37b 0.0029 Phenotypic RFI, kg/d 0.03 ± 0.16 -0.02 0.07 -0.06 ± 0.06 0.79 Genetic RFI, kg/d -0.04 i 0.06 -0.16 0.06 -0.22 ± 0.06 0.59 Dry matter intake, kg/d 10.33 ± 0.25 10.53 ± 0.14 10.55 ± 0.14 0.70 Average daily gain, kg/d 1.36 ± 0.05c 1.48 ± 0.03b 1.50 ± 0.03b 0.039 Metabolic BW, kgO-75 91.28 ± 1.31 91.35 ± 0.84 91.11 ± 0.83 0.93 Feed conversion, kg DM/kg gain 7.96 i 0.23b 7.26 ± 0.12c 7.20 ± 0.12c 0.005 Partial efficiency of growth 0.27 ± 0.009d 0.29 ± 0.004c 0.30 ± 0.003b 0.06 Relative growth rate (x 10.2) 14.8 i 0.59c 16.23 ± 0.25b 16.44 0.24b 0.013 Kleiber ratio, (x 10-2) 1.48 i 0.05c 1.62 i 0.03b 1.72 ± 0.02b 0.013 Ultrasound backfat, mm 7.93 ± 0.55d 8.74 ± 0.23c 9.46 ± 0.21b 0.014 Ultrasound LM area, cm 2 82.20 i 1.42c 84.55 i 0.61b 83.24 0.576c 0.011 Number ofAnimas 26- 143 212 Carcass grade fat, mm 9.52 ± 0.79 10.15 ± 0.36 10.94 ± 0.33 0.10 Average carcass backfat, mm 10.63 ± 0.78d 11.64 ± 0.34c 12.55 i 0.30b 0.039 Carcass LM area, cm 2 84.40 i 1.64bc 85.56 0.81b 83.83 i 0.75c 0.015 Cafcass marbling score 2.45 ± 0.10 2.44 ± 0.05 2.51 ± 0.05 0.47 Lean meat yield 59.13 ± 0.74b 58.56 0.34bc 57.47 i 0.31c 0.024 Carcass yield grade 1.67 0.14 1.59 ± 0.06 1.76 ± 0.06 0.10 a P value from overall F test b, c, d Means in rows followed by different superscripts are different (P < 0.05). Table 6. Association of UASMS2 and A59V genotype combinations with serum leptin concentration, performance, feed efficiency, ultrasound, and carcass merit in crossbred composite cattle. UASMS2 and A59V haplotype Traita. CCCC CCCT CCTT CTCT CITIT TlT SEM Effect, % Pvalueb Animals 31 127 148 47 99 12 SLPT 10.37e 12.91dc 1 4 .10d 14.01d 14.1ld 19.39c 0.80 8.97 <0.001 RFIp 0.02 -0.04 -0.16 0.06 0.07 0.11 0.13 0.13 0.44 RFIg -0.06 -0.19 -0.31 -0.09 -0.07 -0.06 0.13 0.03 0.69 DMI 10.32 10.47 10.48 10.70 10.63 10.91 0.22 0.16 0.29 ADG 1.36d 1.47c 1.51c 1.46c 1.47c 1.51c 0.04 0.87 0.042 MvWr 91.21 91.02 91.04 92.38 91.22 92.12 1.20 0.01 0.70 FCR 7.89c 7.2 4 d 7.08d 7.49dc 7.42dc 7.22d 0.22 0.50 0.019 RGR (x 102) 14.85d 16.18cd 16.54c 15.66cd 15.85d 16.46c 0.40 1.24 0.0028 KRAT (10-2) 1.48d 1.65c 1.64c 1.57cd 1.58cd 1.65c 0.09 1.19 0.0053 UBF 7.82c 8.55de 9.42d 9.16d 9.07d 11.38c 0.43 6.68 0.005 UMAR 5.15e 5.02e 5.11c 5.26de 5.20c 5.55c 0.11 6.26 <0.001 ULMA 82.32d 84.07cd 82.19d 85.71c 82.96d 81.64d 1.13 0.91 0.01 Animals 26 10.9 120 34 84 8 - CGF, mm 9.66c 9.89de 10.89d 10.84d 1 0.55d 13.51c 0.69 5.53 <0.001 CBF, mm 10.75c 11.40de 12.40d 12.27d 12.18d 15.12c 0.70 5.84 <0.001 CMAR 2.42c 2.41c 2
.
4 8de 2.56cd 2 .51cd 2.72c 0.09 4.16 <0.001 CREA 8 4 .33cd 85.16cd 82.60d 86.09c 82.86d 83.70d 1.42 4.85 <0.001 CYG 1.67 1.57 1.74 1.72 1.65 2.04 0.12 4.20 <0.001 LMY 59.07c 58.70cd 57.53d 58.08cd 5 7.80d 55.59e 0.65 8.92 <0.001 I SLPT = serum leptin concentration (ng/mL); RFIp = phenotypic RFI (kg/d); RFIg = genetic RFI (kg/d); DMI 5 daily dry matter intake ((kg/d); MNCfT = metabolic BW (kg.
75 ); ADG = average daily gain (kg/d); FCR = feed WO 2007/129219 PCT/IB2007/001717 46 conversion ratio (kg DM/kg gain); RGR = relative growth rate; KRAT = Kleiber ratio; UBF = ultrasound backfat (mm); UMAR = ultrasound marbling score; ULMA = ultrasound LM area (cm2; CGF = carcass grade fat (mm); CBF = carcass backfat (mm); CMAR = carcass marbling; CLMA = carcass LM area (cm2); CYG = carcass yield grade; LMY = lean meat yield (%). 5 b P values and haplotype effects are from haplotype regression using dummy variables. Haplotype effects are expressed as % of total phenotypic variation in the trait, shown. c, d, c Means in rows followed by different superscripts are different (P < 0.05). Example 3: Ghrelin SNPs This Example illustrates associations between a single nucleotide polymorphism 10 (SNP) in the ghrelin gene with measures of feed intake, growth, and carcass quality in beef cattle. The SNP is a specific A to G nucleotide substitution in intron 3 of the bovine ghrelin gene. Ghrelin is a growth hormone releasing peptide, consisting of 28-amino acids, which serves as an endogenous ligand for growth hormone-secretagogue receptors (GHS-R), which 15 are G-protein-coupled receptors. These receptors in turn stimulate the release of GH from the pituitary gland. In addition to the role of ghrelin in the stimulation of the release of GH, ghrelin also plays a role in the stimulation of appetite and feeding activity through interactions with peptides such as NPY. The experimental animals used in this study were Continental x British hybrid beef 20 steers sired by Angus, Charolais or University of Alberta Hybrid bulls. Feed intake, growth and carcass data were collected over two years under feedlot conditions at the University of Alberta's Kinsella beef cattle research station. Genomic DNA was extracted from blood samples using a standard high salt phenol/chloroform extraction method. Genotyping of the SNP was carried out using the Illumina GoldenGate assay on the BeadStation system 25 (Illumina Inc., San Diego, CA), which allows the simultaneous genotyping of 1,536 SNPs using 250 ng of genomic DNA per sample. The SNP analyzed is an A to G nucleotide substitution in intron 3 of the bovine ghrelin gene (unpublished) and has the sequence is depicted in FIG. 4. Associations of the genotypes for each polymorphism with measures of performance 30 and carcass merit were analyzed using General Linear Mixed Model in SAS. The statistical analyses model included fixed effects of SNP genotype, test year (two levels), contemporary test group nested within year (four levels), breed of sire (three levels), linear covariate of age of animal on test, and random effects of sire and dam of animal. Additive genetic effects were estimated for traits that were or tended to be statistically different (P < 0.10) between 35 different SNP genotypes.
WO 2007/129219 PCT/IB2007/001717 47 Table 7. Genotype and allele frequencies of the Ghrelin SNP in the experimental population of beef cattle. Sire breed Number of Animals AA AG GG G allele frequency Angus 127 97 27 3 0.13 5 Charolais 92 77 14 1 0.09 Hybrid 85 74 11 - 0.06 Total 304 248 52 4 0.10 Table 8. Effect of ghrelin genotypes on performance and carcass merit of beef steers. Ghrelin SNP genotype Trait AA AG P value Number of animals 248 52 Carcass weight, kg 311.10 ± 4.38 298.94 ± 5.90 0.04 Slaughter weight, kg 527.08 ± 7.14 511.78 ± 9.32 0.05 Lean Meat Yield, % 57.86± 0.45 59.10 0.65 0.06 Yield grade 1.60 0.07 1.35 ± 0.12 0.06 Average daily gain, kg/d 1.47 + 0.03 1.39 ± 0.04 0.08 Final weight, kg 460.37 ±6.15 448.16 8.11 0.08 Metabolic mid-weight, kg 7 5 92.76 0.96 91.03 1.25 0.1 Dry matter intake, kg/d 9.42b 0.15 9.16 0.21 0.18 a P values from overall F test. Four animals with genotype GG were excluded from 10 the analyses of ghrelin. Example 4: Neuropeptide Y SNPs This Example illustrates associations between a single nucleotide polymorphism (SNP) in the neuropeptide Y (NPY) gene with measures of growth and carcass quality in beef cattle. The SNP is a specific A to G mutation at the 666 nucleotide position in intron 2 of the 15 bovine NPY gene. Neuropeptide Y (NPY) is a 36-amino acid peptide that plays a powerful role as a central appetite stimulator in the regulation and control of food intake and energy-balance. Neuropeptide Y also stimulates food intake and induces a general anabolic state by reducing energy expenditure. Additionally, NPY influences the regulation of growth in animals by 20 causing a dose-dependent inhibition of GH release, and a lowering of plasma growth hormone and IGF-1 concentration through the stimulation of somatostatin. The experimental animals used in this study were Continental x British hybrid beef steers sired by Angus, Charolais or University of Alberta Hybrid bulls. Feed intake, growth and carcass data were collected over two years under feedlot conditions at the University of 25 Alberta's Kinsella beef cattle research station. Genomic DNA was extracted from blood samples using a standard high salt phenol/chloroform extraction method. Genotyping of the SNP was carried out using the Illumiria GoldenGate assay on the BeadStation system (Illumina Inc., San Diego, CA), which allows the simultaneous genotyping of 1,536 SNPs WO 2007/129219 PCT/IB2007/001717 48 using 250 ng of genomic DNA per sample. The SNP analyzed is an A to G substitution at the 666 nucleotide position in intron 2 of the NPY gene (Accession No. AY491054). The nucleotide sequence is depicted in FIG. 5 (SEQ ID NO: 5. (species, bos taurus): 5 Associations of the genotypes for each polymorphism with measures of performance and carcass merit were analyzed using General Linear Mixed Model in SAS. The statistical analyses model included fixed effects of SNP genotype, test year (two levels), contemporary test group nested within year (four levels), breed of sire (three levels), linear covariate of age of animal on test, and random effects of sire and dam of animal. Additive and non-additive 10 genetic effects were estimated for traits that were or tended to be significant (P < 0.10) between different SNP genotypes. Table 9. Genotype and allele frequencies of the NPY SNP in the experimental population of beef cattle. Sire breed Number of Animals AA AG GG G allele frequency 15 Angus 127 28 61 38 0.54 Charolais 92 18 56 18 0.50 Hybrid 85 9 29 47 0.72 Total 304 55 146 103 0.58 Table 10. Effect of NPY genotypes on performance and carcass merit of beef steers. NPY SNP genotype Trait AA AG GG P value' Number of animals 55 146 103 Ultrasound LM area, cm 2 79.20 ± 0.96 78.55 t 0.61 75.11 L 0.70 0.002 Slaughter weight, kg 540.42 b 8.35 530.66 : 6.67 513.25 d 7.26 0.008 Metabolic mid-weight, kg' 94.31 + 1.14 93.40 ± 0.90 91.29 h 0.99 0.03 Final weight, kg 469.57 = 7.45 464.28 5.89 450.65 + 6.42 0.04 LM area, cm 2 82.74 1.37 82.52 t 1.08 79.63 ±11.19 0.06 Carcass weight, kg 314.63 ± 5.45 311.67+ 4.33 301.19 ± 4.78 0.06 20 a P values from overall F test. Table 11. Additive and non-additive genetic effects of NPY SNP on performance and carcass merit. Dominance Trait Additive Effect P value deviation Pvalue Ultrasound LM area, cm 2 4.09 + 1.19 0.002 -1.39 ± 0.80 0.10 Slaughter weight, kg 27.17 ±48.35 0.004 -3.83 ± 5.48 0.49 Metabolic mid-weight, kg'7 5 3.02 t 1.17 0.02 -0.60 & 0.76 0.44 Final weight, kg 18.92 :17.57 0.02 -4.17 + 4.96 0.41 LM area, cm 2 3.11+1.45 0.05 -1.34 t 0.95 0.19 Carcass weight, kg 13.44 = 5.70 0.04 -3.76 * 3.75 0.34 WO 2007/129219 PCT/IB2007/001717 49 Example 5: Uncoupling Protein 2 SNPs This Example illustrates associations between a single nucleotide polymorphism (SNP) in the uncoupling protein 2 (UCP2) gene with measures of feed intake, growth, and carcass quality in beef cattle. The UCP2 SNP2 polymorphism analyzed is a specific A to G 5 substitution at the 812 nucleotide position in exon 4 of the bovine UCP2 gene (Accession No. XM_614452) and the nucleotide sequence is depicted in FIG. 6 (SEQ ID NO: 6). The UCP2 SNP 1 polymorphism considered is a C to G substitution identified at position 213 in intron 2 of the UCP2 gene according to the following unpublished nucleotide sequence of FIG. 7 (SEQ ID NO: 7). 10 UCP2 has been shown to regulate insulin secretion, and it is up-regulated by a high fat diet, suggesting UCP2 to be important for determining basal metabolic rate and possibly resistance to obesity. Significant geneic linkage has been established between microsatellite markers encompassing the location of UCP2 with resting metabolic rate, body mass, body fatness and fat mass in humans. 15 The experimental animals used in this study were Continental x British hybrid beef steers sired by Angus, Charolais or University of Alberta Hybrid bulls. Feed intake, growth and carcass data were collected over two years under feedlot conditions at the University of Alberta's Kinsella beef cattle research station. Genomic DNA was extracted from blood samples using a standard high salt phenol/chloroform extraction method. Genotyping of the 20 SNP was carried out using the Illumina GoldenGate assay on the BeadStation system (Illumina Inc., San Diego, CA), which allows the simultaneous genotyping of 1,536 SNPs using 250 ng of genomic DNA per sample. Table 12. Genotype and allele frequencies of the UCP2 SNP2 in the experimental population of beef cattle. 25 Sire breed Number of Animals AA AG GG G allele frequency Angus 127 47 63 17 0.38 Charolais 92 46 38 8 0.29 Hybrid 85 48 31 6 0.25 Total 304 141 132 31 0.32 30 Table 13. Genotype and allele frequencies of the UCP2 SNP1 in the experimental population of beef cattle. Sire breed Number of Animals CC CG GG G allele frequency Angus 127 47 62 18 0.39 Charolais 92 46 39 7 0.29 35 Hybrid 85 48 30 7 0.26 Total 304 141 131 32 0.32 WO 2007/129219 PCT/IB2007/001717 50 Table 14. Effect of UCP2 SNP1 genotypes on performance and carcass merit of beef steers. UCP2 SNP1 genotype Trait C _CG GG P-valuea Number of animals 141 131 32 Final weight, kg 455.07 1: 6.91 463.60 ± 6.91 440.94 ± 9.44 0.02 Metabolic mid-weight, kg-" 5 91.97 1.07 93.21 : 1.07 89.79 t 1.45 0.02 Slaughter weight, kg 523.39 7.76 527.41 ± 7.76 506.82 10.67 0.09 Dry matter intake, kg/d 9.37 0.17 9.44 0.17 8.94 0.25 0.11 Lean Meat Yield, % 58.44 0.46 57.61 ± 0.48 58.91 0.79 0.13 Average backfat 10.90 0.36 11.88 t 0.39 10.94 0.81 0.18 Average daily gain, kg/d 1.44 i 0.03 1.48 ± 0.03 1.40 0.05 0.19 Carcass weight, kg 307.47 ± 4.86 311.92 ± 5.01 300.12 t 7.33 0.20 a P values from overall F test. Table 15. Additive and non-additive genetic effects of UCP2 SNP1 on performance and carcass merit Dominance Trait Additive Effect P value deviation P value Final weight, kg 14.13 ± 8.04 0.09 -15.60 1 5.17 0.01 Metabolic mid-weight, kg 7 5 2.18 J 1.22 0.09 -2.32 0.79 0.01 Slaughter weight, kg 16.57+9.15 0.08 -12.31 5.89 0.05 Dry matter intake, kg/d 0.43 h 0.23 0.08 -0.29 i 0.15 0.07 Lean Meat Yield, % -0.47 ± 0.78 0.56 1.07 0.49 0.05 Average backfat -0.05 ± 0.87 0.96 -0.97 0.56 0.11 Average daily gain, kg/d 0.05 ± 0.05 0.35 -0.06 0.03 0.07 Carcass weight, kg 7.34 :h 6.62 0.29 -8.13 4.17 0.08 5 Table 16. Effect of UCP2 SNP2 genotypes on performance and carcass merit of beef steers. UCP2 SNP2 genotype Trait AA AG GG P value' Number of animals 141 132 31 Final weight, kg 455.22 ±6.80 463.76 & 6.80 441.66 9.44 0.02 Metabolic mid-weight, kg' 7 92.01 t 1.05 93.24 i 1.05 89.92 1.45 0.03 Dry matter intake, kg/d 9.37 ± 0.17 9.46 0.17 8.91 * 0.25 0.07 Slaughter weight, kg 523.52+ 7.67 527.54 7.67 507.56 10.70 0.11 Lean Meat Yield, % 58.43 0.47 57.59 ± 0.49 58.82 0.79 0.14 Average daily gain, kg/d 1.44 i 0.03 1.48 i 0.03 1.39 ± 0.05 0.18 Carcass weight, kg 307.49 i 4.82 311.89 = 4.97 301.27 = 7.29 0.25 " P values from overall F test. Table 17. Additive and non-additive genetic effects of UCP2 SNP2 on performance and carcass merit. Trait Additive Effect P value Dominance deviation P value Final weight, kg 13.56 t 8.10 0.11 -15.31 L5.20 0.01 Metabolic mid-weight, kg 7 2.08 A 1.23 0.10 -2.28 ± 0.79 0.01 Dry matter intake, kg/d 0.47 t 0.23 0.06 -0.32 = 0.15 0.05 Slaughter weight, kg 15.96 9.22 0.10 -12.00 5.94 0.06 WO 2007/129219 PCT/IB2007/001717 51 Trait Additive Effect P value Dominance deviation P value Lean Meat Yield, % -0.39 + 0.77 0.63 1.03 0.48 0.06 Average daily gain, kg/d 0.05 10.05 0.32 -0.06 * 0.03 0.07 Carcass weight, kg 6.221 6.60 0.37 -7.51 +4.17 0.10 Example 6 This Example illustrates that there are associations between SNPs and measures of performance and carcass merit in beef cattle. In addition, the additive genetic effect and the dominance deviation of the genotypes was determined. The additive effect is the difference in 5 trait value between the two homozygote genotypes (#AA-$GG). The dominance genotypic value is the deviation of the heterozygote from the mean of the two homozygotes (*AG (*AA + GG)/2). The experimental animals used in this study were derived from a hybrid dam line crossed to Angus, Charolais and hybrid sires. The phenotypic data was collected using a 10 GrowSafe system. SNP genotyping was done using an illumina Beadstation. The SNP genotypes examined in this study were growth hormone receptor (GHR), neuropeptide Y (NPY), Ghrelin, and uncoupling protein-2 (UCP2). Association analysis was conducted in PROC MIXED of SAS with the following variables: (1) fixed effects: SNP genotype, sire breed (three levels), batch nested in year (four 15 levels); (2) random effects: Sire and Dam identification; (3) linear covariate: age at start of test (not used for NPY association). Table 18. Associations of SNP genotypes with performance and carcass merit of beef steers. Trait Gene AA AG GG P-value Average daily gain, kg/d GHR 1.47 1.41 1.65 .004 Ghrelin 1.47 1.39 .08 Dry matter intake, kg/d GHR 9.4 9.3 10.3 .01 UCP2 9.4 9.5 8.9 .07 Carcass LM area, cm 2 GHR 81 81 87 .03 NPY 83 83 80 .06 Ultrasound LM area, cm 2 GHR 77 77 81 .06 NPY 79 79 75 .002 Lean Meat Yield, % Ghrelin 57.9 59.1 .06 Metabolic mid-weight, kg.
75 UCP2 91.9 93.2 89.8 .02 NPY 94.3 93.4 91.3 .03 GHR 91.3 91.3 96.6 .01 Ghrelin 91.0 91.0 .1 Table 19. Additive effect and dominance deviation of the SNP genotypes (effect ± SE) Trait Gene Additive Effect Dominance Deviation Average daily gain, kg/d GHR 0.18 ± 0.07* 0.15 ± 0.04*** Carcass LM area, cm 2 GHR 5.8 ± 2.3* 3.4 ± 1.4* Ultrasound LM area, cm 2 GHR 4.2 ± 1.9* 2.7 ± 1.1* NPY 4.1 ± 1.2** -1.4 ± 0.8 Dry matter intake, kg/d GHR 0.9 ± 0.3** 0.6 ± 0.2** UCP2 0.5 ± 0.2 -0.3 ± 0.2* WO 2007/129219 PCT/IB2007/001717 52 Trait Gene Additive Effect Dominance Deviation Final weight, kg GHR 30 i1** 24 7** NPY 19±7.5* -4i5 UCP2 14 8 -15 5* Metabolic mid-weight, kg' 75 UCP2 2.2 ± 1.22 -2.3 ± 0.79* NPY 3.0± 1.17* -0.6 0.76 GHR 4.0 1.72* 3.3 ± 1.02** *P<0.05, **P<0.01, ***P<0.001 denotes the significance of the effect Table 20. Allele frequency for each gene. Frequency of Alleles Gene A G GHR 0.77 0.23 NPY 0.42 0.58 UCP2 0.68 0.32 Ghrelin 0.9 0.1 FIG. 8 and tables 18-20 demonstrate that the SNP in the GHR gene is associated with body weight, average daily gain, feed intake, and LM area, and has a significant dominance deviation of the A allele over the G allele. In addition, the data show that the NPY gene is 5 associated with body weight and LM area and there is a significant positive additive effect of the AA genotype on these traits. Moreover, the SNP in the UCP2 gene is associated with body weight and shows over-dominance of the heterozygotes. It is also demonstrated that the SNP in the Ghrelin gene shows associations with body weight and average daily gain. Example 9 10 Table 21. Summary of SNPs and Phenotypic Effect in Beef Cattle SNP Effected Carcass Trait I GHR - INT4 REA, QUALITY, CHOICEQG,CALCYG CUTT, MBS, REAHCW, YG 2 UASMS1 COST, FRAME, PREDYG, CARCFAT, HCW, INWT, BFAT, CALCWT, REA,. WT3, CALCYG, CUTT, BFATRATE, MBS, DOF 3 UASMS2 INWT, COST, HCW, ADJRTR, FRAME, CALCWT, WT3, DP 4 UCP2- INT2 HCWVALUE, QUALITY, MBS, CHOICEQG 5 GHREL YG, CALCYG, CUTT, PREDYG, CARCFAT, REA, BFAT 6 NPY- INT2 REA, QUALITY, CHOICEQG, CALCYG, CUTT, MBS, REAHCW, YG Key: ADJRTR = Adjusted Rate of Return to Rancher BFAT = Back Fat BFATRATE = Rate of Deposition of Back Fat 15 CALCWT = Calculated Weight CALCYG = Calculated Yield Grade CARCFAT = Carcass Fat CHOICEQG = % Choice as Measured by Quality Grade WO 2007/129219 PCT/IB2007/001717 53 CUTT = Cutability COST = Cost of gain DOF = Days on Feed DP = Dressing Percentage 5 FRAME = Frame Score HCW = Hot Carcass Weight HCWVALUE = Hot Carcass Weight Value REA = Rib Eye Area INWT = In Weight (weight of animal on arrival at the feedyard_ 10 MBS = Marbling Score QUALITY = Quality Grade PREDYG = Predicted Yield Grade QUALITY = Quality REA = Rib eye area 15 REAHCW = Rib eye area per 100 lbs of hot carcass weight WT3 = Live weight at third weighing period YG = Yield Grade Table 22. Frequencies MARKERS A252T COUNTS GENOT FREQ ALLELE FREQ A252T AA 1367 0.90 0.95 A252T AT 131 0.09 A252T TT 15 0.01 0.05 C963T CC 516 0.33 0.57 C963T CT 757 0.48 C963T TT 306 0.19 0.43 A1457G AA 475 0.30 0.55 A1457G AG 760 0.49 A1457G GG 326 0.21 0.45 FABP4 CC 871 0.55 0.74 FABP4 CG 579 0.37 FABP4 GG 121 0.08 0.26 UASMS1 CC 301 0.19 0.43 UASMS1 CT 764 0.48 UASMS1 TT 512 0.32 0.57 UASMS2 CC 778 0.49 0.70 UASMS2 CT 662 0.42 UASMS2 TT 141 0.09 0.30 EXON2FB CC 510 0.32 0.57 EXON2FB CT 767 0.49 EXON2FB TT 293 0.19 0.43 GHR AA 19 0.01 0.11 GHR AT 304 0.19 GHR TT 1245 0.79 0.89 T945M CC 1391 0.88 0.94 T945M CT 181 0.11 T945M TT 8 0.01 0.06 TFAM1 CC 483 0.31 0.55 TFAM1 CT 729 0.47 TFAMI TT 334 0.22 ,0.45 WO 2007/129219 PCT/IB2007/001717 54 MARKERS A252T COUNTS GENOT FREQ ALLELE FREQ TFAM2 AA 587 0.37 0.60 TFAM2 AC 738 0.47 TFAM2 CC 257 0.16 0.40 TFAM3 CC 486 0.33 0.57 TFAM3 CT 696 0.47 TFAM3 TT 287 0.20 0.43 A59V CC 1161 0.74 0.86 A59V CT 388 0.25 A59V TT 29 0.02 0.14 UCP21NT2 CC 86 0.05 0.23 UCP2INT2 CG 541 0.34 UCP21NT2 GG 950 0.60 0.77 WO 2007/129219 PCT/1B2007/001717 55 on 0%1- r 2tV tl . .0 .0r:I i1:C1 - i C . r- . 00 kn Q0C S0C> 6- (4 < C!~ ' 'R ei I- ci -z - cc rq 1 0 9 - ' 1 C- 'R0 RnO n' ' . 0"t m I 00 .0 C ( -- - - %,0 0p oo, 6M in c- w- -n. b' 6 Nc C> Ccc (4 C > 0 0> w 0= 0 w C) %0 071 CD 0N %0 at0I -m10t C sC 0C ~~~t z~~o ,c r ', cc 4 0 0 M 00 W - ina . ' -- 'o C> cc 0 0 0 C NRC CiC - * - RR C C Ch N l*0 \OC t >4 ~ m >a -m - C )C oC C > C -------------------------- ------------------------------ C 1 0 00 , c C..) C -C 7,oCM oo MCMo1000000 9 9 ,.6 9r a9 ,66 ;* , 7Nm c) 00 as r--t00 IN0CM 0 C14MO O \.o - -a M~ M - D )o CC) CD *o '0 *Ir'nO'n t2.) - - - - - - - - - w3-z 0 C = C ( D C );-r-t I n t 0 0 rj) dQJ 6L a ; C D m o 010' - tnC C)C DC-C 8 ;k 7 - .3- : C DC as4-(,.,rm w m N DC l c- In I ( - IC!I lC C 1c l . . r C C C cni C C C>C )C >C > C
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WO 2007/129219 PCT/1B2007/001717 65 0010 t t t0 k o NC r % nt o- 0U n k 0 0%1 !N , >c >t oo h I- "*W C9o r:r r i . . .C 3 R a t cl C C )c Dc D C C DC >a )C c = = C; o) t q t ); r-0 U C ,t N qc -cN a " C-4N m C oc C- r -rC 7 rCNr 0r -mC c c 0%o * * C14 00 "t 0 O C- %0t q 3 - ":o, 0 0O0 O0 r n q 0 c) m e ON - ke n t O 0 000C % wC- -% m~o cy. o :- m -T W r C% % \ < 00\! -o oo- - e "o 00 C) m00 n0 m ,.,N C4 l0 00 . . .c q0 00... k l- mC " 0 ) 0 )11r nC nr nC >-r r Tt C: 5 C 5 C 5 k 0 -(>Qt UC 1- n C C 60 ;6( ;6C 4e C 6 C'C = >Q C E -cr tn m 00 C in---m-----D------D-- --------------------- C14 11 n t -- -CDoo-m-- - - -- - - -- - -C14 - --- - -- - - -- -- aC=)I 2- cn 00 - ON -Y 0 e -- \0n \0 .0ctC7-o-r 10c~ r- mf r- I' ._ t-n oe Dr n t- en:, cm m - - - ~ -' - H - - ------- WO 2007/129219 PCT/1B2007/001717 66 M h1 n0 1 trm"tN 1 0a 010k tW - 0C 00 r4 o o oM - tTM6=rl=1r-dnM 6 Z o tn .ookn 0oC'4d-"r d o o r-1 U Rll l l R - q c!O iC!0 ON *-00 .04 *0 *)t t0n0 -0 *nM 0 q10C 1 =., O D O -0 M~ kn t.0 en. 0 -: 0 n 0 0 - r M M M- e N O oCN0 C14 1f 00 00 M M. - o 0 0 0 '. '0 (~ 0 0 CD 0 0 0 0 I-C 0~ CN t- 0n -(:r C1 4 CD V$ (= ' r wU M0 (' t-0 - 0tnC 0 M M'o lCC)C \, *. Z0 \* .0 ='o . C1 7 O 0 0O C!C = l a ! Ci q C l l nI 10C ~ lC ) C )C= C DC : ON a, D M o0 t<,C4r -M 0 1 -t -( rt C C 10 c W)00 d o o o - o oKr t roCD C \D6q C o o oo Vq0Dq = D< >C4C I I 7 C!Ci r RC I 0,6a ,6C 6 a 6( - = D-r--rMm-q , 5c 0a000 N- 'It r M0 0 M C1 %D 0. l en. 004* -- 0 wN 000 4 N -4 0 q *004 \o0 I0 0o - (' CR C:6 C) . q. C! 0 C! - CO 4 ta CR CR q CD 0 a- a a a a 0 0 6 6 C 9O 9 : T9 ? o : c00 tn M k nt ) qM t r C nr -\ M " O : i- -CC - " M - n mC)C 1 o a - as' I- Et qk O -N'c WO 2007/129219 PCT/1B2007/001717 67 c) Q 6 ) 00 c' cc c * 0o 00 %D .%0%3 %CC) cnC (c) D CO c00 - C1 r C11 e00C m C) 00 -Itt-W) 000 O0 CO -o6 i- r ~ - _ cri M- CD -. t- 4 0. C) N CD o o *D m. -- cn -n 0% ONal CD CD ~ ~ -( c ( Q Cot c 0% cc 0.000(O ol r- C>-~ %0 t *I - N0rNC-- - CD " 00 (N mO ON cc ( t- (0 C-- - cq C% ON CD- =) \. ccrO-( 0 f C kflC - CN (Nj - h0 0C4ON10t 01:1 Do \o N 0 r- t '0 00 00 , q0 00 0%%CC 0C) 0O oo Inr- q 00C)00OOOO C4 %)Cm)00)n mt--)-- 10 r- C)CZ- O0(N%0 N nV 5u cc -5 mn C-4 C4 t- ', r-0 "oo 'A ON Sc C-4 C-- t-- C>. 'n 'm. r, m C-D w~ CC- 0 c, ~L~~( )C)o - i > ~ ~ ~ ~ - ' -~ CD C)a*C w m \ 0ID C) m 1. . . . . . . .C) 1 r. 'n o o O9a! . C C>C >C DC )C >C > C DC D D C D=C )C .c>C >- = m 0- 4 C4 N C4 C1 C q C4 N3iC4 C1CCC,;r C1 0C4I nm m m nme Fa.Cun w n W) t n k Cu wl wl "1 wl z N %%%000 C> 0% N Tr )%C\%n m0\0%0 m0% w C, C 'o \ 't-(5cr t- n n ) 4 0 ~r 0 . " q* N c m m \ Cc c )q 0 r ,:Ci' co ocD D- -mr- C D DCw >C , L- !L- WO 2007/129219 PCT/1B2007/001717 68 4= r- m. en - c c - -n 2o r- -0 ' 0% m~ m c N c CD 0 Ch > mON%0r-t 0 - C14 0%0%0%N- Ch -M 3 00 00 me e o 000000-00-0u =0"--nCn - C M (D -m w CD t0 -m -%D t- C14 .4- C5c c C nC r! C'i N C'!w V 0~ r- t R Rq. . . cl! % C> > >C> C C D ~r. CM C0 *e (Dcl C *CD-o0 4 )4 =C 10 r. M- -, CM I' MC - w T mf t- vt %D. 0D r% - C 0 Du 00 m 0 n 0 0 -h--- m M Lo oq RRC= 'I l ! C chmcn6C566 )m ,6 D4=oo DCDc C C 6 C C % Dm 0 = m ON0 00 0 0 0- C C tn<1 ~aad "r 0 .0 00 %nenklo :tC4 > -- D :) r C= m m C4 i" s % go C>-en ---------------- Q C5 6 .0%000e0%c cc--O Oc0 Cc l= CO - 00 0 \0 ) tn!0tN'D m C a, , 6 6 6 CD 6 - d c. 0 ci* =- 6~ c, o 6% C) CD 0 0 00 C M0 0 %
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% 0 0 ON 00knc~eC -1 m- .0 1.0 *CtMOl D > C C c>C C~ m -N;;i 1 DC C 00 -100 tn ~~ ~ ~Q tn knCDrq" n WO 2007/129219 PCT/1B2007/001717 69 m r\ D 0 t"~ N C oIDk 0- ot N >. C1 l > = C RCiI Co -c- -: a, , n % . o i .4- = 00 = N P CD 9O 9 9 o C 6CC 0 w * h DC 00 0 00 0 00 0 00 0 :; 0) Nr r- ON 0,0 w 0 C14 O\ m .C- r- r- - qr f ) , - zr- w- - m 00 t- tC0 C5) 6. CD 6 6 6 6C)c -c>C Dc ,c >c, b 6 = Z : R s t-t,(: r m 0 dr-e = *CNe .n( n. ,c 1o.0 00C no o0 0 a c . 0 02"t 1 CDT 5 C 0w\DO 3) mC N D C>eq'r O CI ;6 C 03>C ) )C ) C D= >C w 0(/m ), o 2 NC na\m10C,0 iQ -:C , mk lr D\ -r-a = m10(4%0"0 St-w0 cl n\q\q w q . Q O Z OQ ( !C C l iQ 1 1: iN WO 2007/129219 PCT/1B2007/001717 70 %0 t , 0 l 1.1t - N k7, 0 r o r . Nt >1 oooo In 0 C6 q 09d00C6oCS n Cdi n en 0 C.< CC DC)C> C 0c o cc ~ 6 o~ CD 66d'r mtc-I 00 -T -000 k \ 0 o E "m !"4 0 D" 5 r ,= -c n1 \=-C ;tnC 0u n100 . Zn* iC C D r ) oC ) C5 ~-- - CD (DC ?mg \.6 dd 6 ~ do odD -%C t rOe006in-n666em m n0%0 F4Cim C4o - tCmt-"?C4 0 r W \ n C n O )r Cz C .,CD=C C -r r- M M~ M m Cc q -Iq C1 C> ~ C') w ~ 01 CO 0 ON(1(7r C- C, C> cn CD0~ -0c W c
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666n6n5 , C C \0 0 0d66C6%00 o0006N6 00%D00 _ -e' e~c~e~' (4 -(' ~4(' e~e~ CD OC CC ~ O -= CO0 C-- 00 CD C ) C=) c - L - ----- --- w----- m---4------------------>---------------------------------------- C> 00 00 m 00 kzj i n t- en C - - t.C r-oCS tn m t, *4 nr 1 en .0...................0 0-0 "o "a % io CD %00 It CONTm2 r! Co06 O0 oo.o.o r aC '4 .0. C, C -- C a ----------------------------------------------------------------------------------- CDI-C l nr e -- "-%0 N o"C4C4e lO E r lC) 4,a Dw rC ON C D-rV- 1c ) ol - % 1 t--C4C D N CuC a.a -A A F - -0 -IP <U g -<<g 0. 4 . . 4 W - - . . n n 40rnCe 0mw wc r W0 n n n > -~ WO 2007/129219 PCT/1B2007/001717 71 in lr m -Ir \ ,r 0 0 m " rC-(," r -k 0 \0 00o q 00 -1 q - no n - n 00en W) - Trtoo o o 0o n IlrO N ON 0 %0 C o)M M< hUit G N;;;;m c I~ . a..R"tC C it l ol 9- l 4 CD ClC)C C D< )( o = 6 C )6 C 0 00r 00 ooC) ww cz: - 00 r'q '. 1 q 0% e 04 I'D C= m~ m- mr wr ON 00 C) cl m'. C14 C> - ~ ri 0 m %5J ;s m0 =r *- *% m. =~ 0- 0- 0 r- tn2 caa 00 m~ m r-I -=4 'IO M10 CD C7'.000-t n Dt-Ca .0t 00 000mc C)000C >I' DC 00O r400 S:400 < C O O %0 c)20 c= w 'DC- c CDC or r ,~ Q 00 00 0,- CD <DC00000 C D(DC ) D t_ g r C:, C.0.000 M tn m~ cc Ir iir0%0 l- 1.0 '.D (cn' ON 00 '.1 00 tn %70 % -- 0 0% cc ('.1 In co 0 C14 kn C4a C1 M% %D 110 %o10% nO ' 0 0 nm oc 0 r -wO - l %6oo 6 do " N0 W 00 0 0 0t-000 > w w I' 00 - 3. = n n 0 0 0 0 tn a%- o"J tn~ CD00 , r 00 ON-----M ------ 0 -- --- - - - - - 00l C1 O 0 n000 cz) Cl00 C = k 40 C 6- q: WO 2007/129219 PCT/1B2007/001717 72 ko 0 tnC4CS o 'Den % N 0 t r-%DD O0 Coe0 0 z lw m 0 0 00i el- r- 0 0 w -.- tnc z k 4m o cs e-no~ " 00 r-~ Tr Q m 00 ; . m , q00 0 r4 00 %-- 0 - 4 C4 a' cI ~\Oa Cl C- cl 0o "0 co tr C'I IC- t- 00 C - eq t- tn in 5 - YNQ QrONen clC 0 N 0 0c o, n 4 '0" e400 mQ, 4,%n. 10~ in- C5 w' -0 C: = " -:-: c!I C4 '-If ~ 00 kn CIt C,4 "- 000 "ol m. kn 0 00 ON %.D ON '.0 0- %na l - M E~~ ~~ %J~ =r :;W en~ C14 I- I' a C 00 00 - - - - - - - - - - - - - - - - - - - -::: -- - - - : , -- -=-- ci ~ m r 0 00 %0 Nr rn a ON) %0 0n tn 1 .a cq tn 0 00 e C'I - f - - - - - - - - - - - - - - - - - - - - - - - - A nO n00C.D C400t-N- 0 mmW n0 I- - 1 i 1q I l o e R C C:) c)C c Q() c o c 0C: 0 C >i >C C = .~ 0 m " 1 cfl " ~ - G Cb a,% Q~~~~~ ~ ~ ~ CD. <7r.L.LMILL) hC =C ' O %C7 %M M NMO C' ; * 1 en << n -n t COD ~ o~ ~ 01 nQwON'Tm 'm r4r ' -r N t-Mt qQ C!IR C! q !InJio> I
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WO 2007/129219 PCT/1B2007/001717 73 Cr ,C ,C 3 0 t . 3C ,z '* C = R'C 0 0 C>ClM W1DC 0% 09RO l( cz C C 6C )C DC )C DC >C : DC D4 . rDo r-0o r4 - C D "D 0 D 00V < m R mI'00C<5 G r 8 <Z> - D C D gO0 CO, , ' CD8,C SC C_ C N0.C5c %0..2 01 >,ch ~ 4 *r-Oltl 0C O -0 0 0 00 0 0 0 0% 0 0 --------- 00" n "tc l - % c -e 0=k m Ce z lc - 0 N -= , " 8 r - D CD Q -8 01 =lclm C qC w ,r 7 n o m W l ID.0 t n C4Q r C-4 m o m k-0n0 io 000 00 0 0\ 000<=o oo' CD C ~ ~ . '" *' r* *" Z- 4- , .0 4- C)O .00 * O C (O 00F- C> 4 1 8 0 C' -- IO cc I lqc!qC rx.q C R \ WO 2007/129219 PCT/1B2007/001717 74 C~ lM' nC * -V 0M 0 (-0 -O .rO C ' nO r- Mk )e -c 00 Dt nc I nk n10 I ne C C>C>00 _n kn% _0 %Nr \0 - oc V, -n r- _o .. 14 m 0 m _ _ I C- ; n I ON E'- t- C> % 00= - o. C0 inn0 *l %. V 02 C>O "0 * a% C> *00 .0 0000 00 m N r G -: q ,r 00 It r- Cl 0 0 - * -0 ., c rf4 0%c- t'- In 0- 0n r. mI0 I 00 0 cq 00 0 . N. 0 0%C1 (W4%or nt s \DCI- " w w r 1 40n0 CiC C c! O In I-N 0Nqc .C'..le' . . . 0%riCi ei 0-0e c "o m.00 %0 100C0 0l - It n N I 0'01*-T ko10 It m > n w.~ CD 'IT r-~ kn cn0%I en M" 0m C4~ rn mn =-, rn e=0N 4 C!In In *C - r 0-0
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C- G -Q> CJ cNr WO 2007/129219 PCT/1B2007/001717 75 cu . l =1o d ~ ~ d L.' Ch 9 0 %0 " CD o -T , q !c m ti6 0i6 oi6 - C ( W~ C J Wr C- "t mO0.0 0 0 -r c.0 csc c c =0 %0C =t 00 ' nr %0 00r l C D c Z o Z - Z v RC R:: 3-. - - 6 N6 -CIC )( : WO 2007/129219 PCT/IB2007/001717 76 Example 8 FIG. 9 shows a flowchart of the input of data and the output of results from the analysis and correlation of the data pertaining to the breeding, veterinarian histories and performance requirements of a group of animals such as from bovines. The flowchart 5 illustrated in FIG. 9 further indicate the interactive flow of data from the computer-assisted device to a body of students learning the use of the method of the invention and the correlation of such interactive data to present an output as a pie-chart indicating the progress of the class. The flowchart. further indicates modifications of the method of the invention in accordance with the information received from the students to advance the teaching process 10 or optimize the method to satisfy the needs of the students. FIG. 10 illustrates potential relationships between the data elements to be entered into the system. Unidirectional arrows indicate, for example, that a house or shed is typically owned by only one farm, whereas a farm may own several houses or sheds. Similarly, a prescription may include have several veterinarian products. 15 FIG. 11 A illustrates the flow of events in the use of the portable computer-based system for data entry on the breeding and rearing of a herd of cows. FIG. 11B illustrates the flow of events through the sub-routines related to data entry concerning farm management. FIG. 11 C illustrates the flow of events through the sub-routines related to data entry concerning data specific to a company. 20 FIG. 12 illustrates a flow chart of the input of data and the output of results from the analysis and the correlation of the data pertaining to the breeding, veterinarian histories, and performance requirements of a group of animals. The invention is further described by the following numbered paragraphs: 1. A method for sub grouping animals according to genotype wherein the 25 animals. of each sub-group have a similar polymorphism in a GHR, ghrelin, leptin, NPY or UCP2 gene comprising: (a) determining the genotype of each animal to be subgrouped by determining the presence of a single nucleotide polymorphism in the GHR, ghrelin, leptin, NPY or UCP2 gene, and 30 (b) segregating individual animals into sub-groups wherein each animal in a subgroup has a similar polymorphism in the GHR, ghrelin, leptin, NPY or UCP2 gene. 2. A method for sub grouping animals according to genotype wherein the animals of each sub-group have a similar genotype in the GHR, ghrelin, leptin, NPY or UCP2 gene comprising: WO 2007/129219 PCT/IB2007/001717 77 (a) determining the genotype of each animal to be subgrouped by determining the presence of a single nucleotide polymorphism(s) of interest in the GHR, ghrelin, leptin, NPY or UCP2 gene, (b) segregating individual animals into sub-groups depending on whether the animals 5 have, or do not have, the single nucleotide polymorphism(s) of interest in the GHR, ghrelin, leptin, NPY or UCP2 gene. 3. The method of paragraphs 1 or 2, wherein the single nucleotide polymorphism(s) of interest is selected from the group consisting of an A to G substitution at the 300 nucleotide position in intron 4 of the GHR gene, an A to G substitution at position 10 212 in intron 3 of the ghrelin gene, a C to T mutation at position 528 in the leptin gene, a C to T mutation at position 321 in the leptin gene, an A to G substitution at the 666 nucleotide position in intron 2 of the NPY gene, an A to G substitution at position 812 of exon 4 in the UCP2 gene and a C to G substitution at position 213 in intron 2 of the UCP2 gene. 4. A method for sub grouping animals according to genotype wherein the 15 animals of each sub-group have a similar genotype in the GHR, ghrelin, leptin, NPY or UCP2 gene comprising: (a) determining the genotype of each animal to be subgrouped by determining the presence of an A to G substitution at the 300 nucleotide position in intron 4 of the GHR gene, an A to G substitution at position 212 in intron 3 of the ghrelin gene, a C to T mutation at 20 position 528 in the leptin gene, a C to T mutation at position 321 in the leptin gene, an A to G substitution at the 666 nucleotide position in intron 2 of the NPY gene, an A to G substitution at position 812 of exon 4 in the UCP2 gene or a C to G substitution at position 213 in intron 2 of the UCP2 gene, and (b) segregating individual animals into sub-groups depending on whether the animals 25 have, or do not have, an A to G substitution at the 300 nucleotide position in intron 4 of the GHR gene, an A to G substitution at position 212 in intron 3 of the ghrelin gene, a C to T mutation at position 528 in the leptin gene, a C to T mutation at position 321 in the leptin gene, an A to G substitution at the 666 nucleotide position in intron 2 of the NPY gene, an A to G substitution at position 812 of exon 4 in the UCP2 gene or a C to G substitution at 30 position 213 in intron 2 of the UCP2 gene single nucleotide polymorphism in the GHR, ghrelin, leptin, NPY or UCP2 gene. 5. A method for identifying an animal having a desirable phenotype relating to certain feed intake, growth rate, body weight, carcass merit and composition, and milk yield, as compared to the general population of animals of that species, comprising determining the WO 2007/129219 PCT/IB2007/001717 78 presence of a single nucleotide polymorphism in the GHR, ghrelin, leptin, NPY or UCP2 gene of the animal, wherein the polymorphism is selected from the group consisting of an A to G substitution at the 300 nucleotide position in intron 4 of the GHR gene, an A to G substitution at position 212 in intron 3 of the ghrelin gene, a C to T mutation at position 528 5 in the leptin gene, a C to T mutation at position 321 in the leptin gene, an A to G substitution at the 666 nucleotide position in intron 2 of the NPY gene, an A to G substitution at position 812 of exon 4 in the UCP2 gene or a C to G substitution at position 213 in intron 2 of the UCP2 gene, wherein the presence of either an A to G substitution at the 300 nucleotide position in intron 4 of the GHR gene, an A to G substitution in intron 3 of the ghrelin gene, a 10 C to T mutation at position 528 in the leptin gene, a C to T mutation at position 321 in the leptin gene, an A to G substitution at the 666 nucleotide position in intron 2 of the NPY gene, an A to G substitution at position 812 of exon 4 in the UCP2 gene or a C to G substitution at positiori 213 in intron 2 of the UCP2 gene single nucleotide polymorphism is indicative of a desirable phenotype relating to certain feed intake, growth rate, body weight, carcass merit 15 and composition, and milk yield. 6. The method of any one of paragraphs 1 to 5 wherein the animal is a bovine. 7. The method of any one of paragraphs I to 7 wherein the GHR, ghrelin, leptin, NPY orUCP2 gene is a bovine GHR, ghrelin, leptin, NPY or UCP2 gene. 8. An interactive computer-assisted method for tracking the rearing of livestock 20 bovines comprising, using a computer system comprising a programmed computer comprising a processor, a data storage system, an input device, an output device, and an interactive device, the steps of: (a) inputting into the programmed computer through the input device data comprising a breeding history of a bovine or herd of bovines, (b) inputting into the programmed computer through the input device data comprising a veterinary history of a 25 bovine or herd of bovines, (c) correlating the veterinary data with the breeding history of the bovine or herd of bovinesusing the processor and the data storage system, and (d) outputting to the output device the breeding history and the veterinary history of the bovine or herd of bovines. 9. The method according to paragraph 8, wherein the computer system is an 30 interactive system whereby modifications to the output of the computer-assisted method may be correlated according to the input from the interactive device . 10. The method according to paragraph 8, further comprising the steps of inputting into the programmed computer diagnostic data related to the health of the cow or herd of cows; and correlating the diagnostic data to the breeding and veterinary histories of WO 2007/129219 PCT/IB2007/001717 79 the cow or herd of cows. i1. The method according to paragraph 8, wherein the veterinary data comprises a vaccination record for a cow or herd of cows. 12. The method according to paragraph 10 wherein the health data is selected 5 from the group consisting of husbandry condition data, herd history, and food safety data. 13. The method according to paragraph 8, further comprising at least one further step selected from the group consisting of inputting into the programmed computer data related to the quality control of the bovine or herd of bovines and correlating the quality control data to the breeding and veterinary histories of the cow or herd of cows, inputting into 10 the programmed computer performance parameters of the cow or herd of cows; and correlating the required performance parameters of the bovine or herd of bovines to a specific performance requirement of a customer, correlating the vaccine data to the performance parameters of the bovine or herd of bovines, correlating herd to the performance parameters of the bovine or herd of bovines, correlating the food safety data to the performance 15 parameters of the bovine or herd of bovines, correlating the husbandry condition data to the performance parameters of the bovine or herd of bovines, inputting into the programmed computer data related to the nutritional data of the bovine or herd of bovines; and correlating the nutritional data to the performance parameters of the bovine or herd of bovines, and alerting to undesirable changes in the performance parameters of the bovine or herd of 20 bovines. 14. The method according to paragraph 8, further comprising the steps of inputting into the programmed computer through the input device data comprising a genotype of a bovine; correlating a physical characteristic predicted by the genotype using the processor and the data storage system; and outputting to the output device the physical 25 characteristic correlated to the genotype for a bovine or population of bovines, and feeding the animal(s) a diet based upon the physical characteristic, thereby improving bovine production. 15. The computer-assisted method according to paragraph 8 for optimizing efficiency of feed lots for livestock comprising outputting to the output device the breeding 30 and veterinary history of the bovine or herd of bovines and feeding the animal(s) a diet based upon their breeding and veterinary histories, thereby optimizing efficiency of feed lots for the bovine or herd of bovines. 16. A method of transmitting data comprising transmission of information from such methods according to paragraph 8, selected from the group consisting of WO 2007/129219 PCT/IB2007/001717 80 telecommunication, telephone, video conference, mass communication, a presentation, a computer presentation, a POWERPOINTTM presentation, internet, email, and documentary communication. 17. An interactive computer system according to paragraph 8 for tracking 5 breeding and welfare histories of poultry comprising breeding and veterinarian data corresponding to a bovine or herd of bovines, and wherein the computer system is configured to allow the operator thereof to exchange data with the device or a remote database. 18. The interactive computer system according to paragraph 17, wherein the input and output devices are a personal digital assistant or a pocket computer. 10 19. A method of doing business for tracking breeding and welfare histories of livestock comprising breeding and veterinarian data corresponding to one or more livestock animals comprising providing to a user the computer system of paragraph 17. 20. A method of doing business for tracking breeding and welfare histories of livestock comprising breeding and veterinarian data corresponding to one or more livestock 15 animals comprising providing to a user the computer system of paragraph 18. 21. The method of doing business according to paragraph 19, further comprising providing the animal owner or customer with sample collection equipment, such as swabs and vials useful for collecting samples from which genetic data may be obtained, and wherein the vials are optionally packaged in a container which is encoded with identifying indicia. 20 22. The method of doing business according to paragraph 8, wherein the computer system further comprises a plurality of interactive devices and wherein the method further comprises the steps of a receiving data from the interactive devices, compiling the data , outputting the data to indicate the response of a student or class of students to a question relating to the operation of the computer-assisted method, and optionally modifying the 25 operation of the computer-assisted method in accordance with the indication of the response. 23. The method of any one of paragraphs 8 to 22 wherein the data comprises presence or absence of one or more of a single nucleotide polymorphism(s) of interest in the GHR, ghrelin, leptin, NPY or UCP2 gene. 24. The method of paragraph 23 wherein the single nucleotide polymorphism(s) of 30 interest is selected from the group consisting of an A to G substitution at the 300 nucleotide position in intron 4 of the GHR gene, an A to G substitution at position 212 in intron 3 of the ghrelin gene, a C to T mutation at position 528 in the leptin gene, a C to T mutation at position 321 in the leptin gene, an A to G substitution at the 666 nucleotide position in intron 2 of the NPY gene, an A to G substitution at position 812 of exon 4 in the UCP2 gene and a WO 2007/129219 PCT/IB2007/001717 81 C to G substitution at position 213 in intron 2 of the UCP2 gene. Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to 5 particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims (3)

1. A method for identifying a bovine animal having a higher average daily gain (ADG), a higher final weight (FW), a higher dry matter intake (DMI), a higher 5 metabolic mid-weight (MMW), a higher slaughter weight (SW), a higher loin muscle area (LMA), a higher ultrasound loin muscle area (ULMA), and a higher feed conversion ratio (FCR) as compared to a general population of bovine animals, comprising the steps of: (a) obtaining a biological sample from said bovine animal, wherein the 10 sample comprises nucleic acids comprising the bovine growth hormone receptor (GHR) gene; (b) detecting in said nucleic acids the presence of a single nucleotide polymorphism (SNP) in the GHR gene, wherein the SNP is a G in both alleles of the GHR gene at position corresponding to position 300 of SEQ ID NO:1; and 15 (c) correlating the G in both alleles of the GHR gene to higher FW, DMI, MMW, SW, LMA, ULMA, and FCR, thereby identifying said bovine animal.
2. A method for producing a population of bovine animals having a higher number of offspring with a higher average daily gain (ADG), a higher final weight (FW), a 20 higher dry matter intake (DMI), a higher metabolic mid-weight (MMW), a higher slaughter weight (SW), a higher loin muscle area (LMA), a higher ultrasound loin muscle area (ULMA), and a higher feed conversion ratio (FCR), as compared to a general population of bovine animals, comprising the steps of: (a) obtaining a biological sample from each bovine animal from a general 25 population of bovine animals, wherein the sample comprises nucleic acids comprising the bovine growth hormone receptor (GHR) gene; (b) detecting in said nucleic acids the presence of a single nucleotide polymorphism (SNP) in the GHR gene, wherein the SNP is a G in both alleles of the GHR gene at position corresponding to position 300 of SEQ ID NO:]; 30 (c) segregating individual bovine animals into sub-groups depending on whether the animals have, or do not have, a G in either or both alleles of the GHR gene at position corresponding to position 300 of SEQ ID NO: 1; (d) continuously breeding only animals that are heterozygous or homozygous for the G in the GHR gene at position corresponding to position 300 of SEQ ID NO: 1, 35 thereby producing the population of bovine animals.
3. The method of claim I or claim 2, substantially as hereinbefore described.
AU2007246772A 2006-01-13 2007-01-16 Polymorphisms in growth hormone receptor, ghrelin, leptin, neuropeptide Y, and uncoupling protein 2 genes and their associations with measures of performance and carcass merit in beef cattle Ceased AU2007246772B2 (en)

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